Amino acid sequence variations resulting from single-nucleotide polymorphisms (SNPs) were identified using a novel mass spectrometric method. This method obtains 99+% protein sequence coverage for human hemoglobin in a single LC-microspray tandem mass spectrometry (microLC-MS/MS) experiment. Tandem mass spectrometry data was analyzed using a modified version of the computer program SEQUEST to identify the sequence variations. Conditions of sample preparation, chromatographic separation, and data collection were optimized to correctly identify amino acid changes in six variants of human hemoglobin (Hb C, Hb E, Hb D-Los Angeles, Hb G-Philadelphia, Hb Hope, and Hb S). Hemoglobin proteins were isolated and purified, dehemed, (S)-carboxyami-domethylated, and then subjected to a combination proteolytic digestion to obtain a complex peptide mixture with multiple overlaps in sequence. Reversed-phase chromatographic separation of peptides was achieved on-line with MS utilizing a robust fritless microelectrospray interface. Tandem mass spectrometry was performed on an ion trap mass spectrometer using automated data-dependent MS/MS procedures. Tandem mass spectra were collected from the five most abundant ions in each scan using dynamic and isotopic exclusion to minimize redundancy. The spectra were analyzed by a version of the SEQUEST algorithm modified to identify amino acid substations resulting from SNPs.

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SUMMARY 1. The elctrophoretic pattern of phosphohexose isomerase has been examine in the blood of 3397 unrelated individuals from several different populations groups. 2. Eight variant phenotypes were identinfied and these were designated PHI 2‐1, 3‐1, 4‐1, 5‐11 6‐1, 7‐1, 8‐1, 9‐1. All of these were rare in the populations studied except the variant designated PHI 3‐1, which was observed with a frequency of about 1% in a mixed population of Asiatic Indians. 3. Studies of selected families indicated that the variants occured in individuals who are heterozygous for one or another of a series of rare alleles at an autosomal locus. 4. Studies on the family of a patient that the patient was heterozygous for two different rare alleles at the PHI locus, each associated with reduced PHI activity. The patinet's mother showed the PHI 9‐1 phenotype and his father showed a new phenotype designated PHI 10‐1. The patient's phenotype has been designeated PHI 9‐10. 5. The enzyme appears to be a dimer and in heterozygotes isozymes of hybrid submit compostion as well as isozymes of like subunit compostion are apparently formed.

SUMMARY We report two families in which a non-progressive spinocerebellar syndrome and a sideroblastic anaemia are segregating together in an X linked recessive fashion. Four males in two generations of one family and a fifth male from an unrelated family had both conditions. Both the sideroblastic anaemia and the spinocerebellar syndrome differ from those which have previously been reported to be inherited in an X linked recessive manner. The association of these two clinically distinct disorders in two unrelated families suggests that they are either two closely linked loci which have undergone simultaneous mutation or pleiotropic effects of an altered allele at a single locus.All the heterozygous women had normal neurological examinations and normal haematocrits and red cell indices. Some had ring sideroblasts on bone marrow examination, a dimorphic peripheral blood smear, and raised serum free erythrocyte protoporphyrin, suggesting that a proportion of heterozygotes can be detected by appropriate haematological studies.We report two families in which a sideroblastic anaemia and spinocerebellar syndrome are segregating together in an X linked recessive fashion. Affected males have a moderate hypochromic, microcytic anaemia with ring sideroblasts on bone marrow examination. Although similar to the previously described X linked hereditary sideroblastic anaemia, this disorder is characterised by raised, rather than normal or low, free erythrocyte protoporphyrin levels and by a lack of excessive parenchymal iron storage in adulthood. The spinocerebellar disorder is different from the other reported X linked ataxias. It is characterised by a non-progressive ataxia and incoordination, as well as long motor tract signs in the younger affected boys. Intelligence is normal. Some heterozygous females have ring sideroblasts on bone marrow examination, a dimorphic peripheral blood smear, and raised serum free erythrocyte protoporphyrin. The proband (patient IV.5) was a 51/2 year old white Received for publication 15 February 1984. Accepted for publication 2 August 1984. male born at term, weighing 3-1 kg. At one year of age he had developmental delays because of truncal ataxia, dysmetria, and tremulousness. At 21/2 years of age, when admitted to hospital for an unrelated illness, he was found to be anaemic (table 1). Peripheral blood smear showed microcytic, hypochromic red cells with anisocytosis and cigarshaped cells. He had no hepatosplenomegaly. Bone General physical examination was normal including height (83-0 cm) and OFC (49 cm). He had marked ataxia while sitting which usually required him to assume a tripod position, marked dysmetria, pathologically brisk deep tendon reflexes in his lower extremities, and a left extensor plantar response. Muscle tone was normal. He had no nystagmus or strabismus.Three males (IV.4, IV.6, and IV.7) who were normal neurologically had normal haematocrits, haemoglobin levels, red cell indices, and peripheral smears.The obligate heterozygote women (11. 1, 111.2, III.6, and II1.7...

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