F. Giannelli

Haemophilia B: database of point mutations and short additions and deletions, 7th edition

The seventh edition of the haemophilia B database lists in easily accessible form all known factor IX mutations due to small changes (base substitutions and short additions and/or deletions of <30 bp) identified in haemophilia B patients. The 1535 patient entries are ordered by the nucleotide number of their mutation. Where known, details are given on: factor IX activity, factor IX antigen in circulation, presence of inhibitor and origin of mutation. References to published mutations are given and the laboratories generating the data are indicated.

Haemophilia B: Database of point mutations and short additions and deletions—eighth edition

The eighth edition of the haemophilia B database (http://www.umds.ac. uk/molgen/haemBdatabase.htm ) lists in an easily accessible form all known factor IX mutations due to small changes (base substitutions and short additions and/or deletions of <30 bp) identified in haemophilia B patients. The 1713 patient entries are ordered by the nucleotide number of their mutation. Where known, details are given on: factor IX activity, factor IX antigen in circulation, presence of inhibitor and origin of mutation. References to published mutations are given and the laboratories generating the data are indicated.

Construction of a 2.6-Mb contig in yeast artificial chromosomes spanning the human dystrophin gene using an STS-based approach

A sequence tagged site (STS)-based approach has been used to construct a 2.6-Mb contig in yeast artificial chromosomes (YACs) spanning the human dystrophin gene. Twenty-seven STSs were used to identify and overlap 34 YAC clones. A DNA fingerprint of each clone produced by direct Alu-PCR amplification of YAC colonies and the isolation of YAC insert ends by vectorette PCR were used to detect overlaps in intron 1 (280 kb) where no DNA sequence data were available, thereby achieving closure of the map. This study has evaluated methods for mapping large regions of the X chromosome and provides a valuable resource of the dystrophin gene in cloned form for detailed analysis of gene structure and function in the future.

Cytogenetics of Down’s Syndrome (Mongolism) I. Data on a Consecutive Series of Patients Referred for Genetic Counselling and Diagnosis

The chromosome findings in a series of 173 Down’s syndrome or presumptive Down’s syndrome index patients, and a further 3 5 pairs of parents of Down’s syndrome index cases, are given. One hundred and forty-four index cases had primary G21 trisomy; six were interchange trisomics, six were mosaics, and one was a chromatin positive male with XXY sex chromosomes and primary G21 trisomy. Sixteen index patients whose diagnosis could not be clinically confirmed, were found to have normal chromosomes. The 35 pairs of parents all had normal chromosomes. In seven families (three index patients with primary G21 trisomy, two with normal chromosomes and two index patients not studied) minor morphological chromosome anomalies were observed. The relevance of this data to the causal association of Down’s syndrome and G21 trisomy, the frequency of mosaicism, double trisomy and association with other chromosome anomalies, is discussed.

Factor VIII gene explains all cases of haemophilia A

Using an mRNA-based method to examine haemophilia A mutations we provide an explanation for the puzzling report that half of the mutations causing severe disease are not detected by analysis of the putative promoter, exons, and most exon/intron boundaries of the factor VIII gene. An unusual cluster of mutations involving regions of intron 22 not examined earlier leads to defective joining of exons 22 and 23 in the mRNA and caused haemophilia A in 10/24 severely affected UK patients.

Int22h‐related inversions causing hemophilia A: a novel insight into their origin and a new more discriminant PCR test for their detection

Summary.  Background: Intrachromosomal, homologous recombination of the duplicon int22h‐1 with int22h‐2 or int22h‐3 causes inversions accounting for 45% of severe hemophilia A, hence the belief that int22h‐2 and int22h‐3 are in opposite orientation to int22h‐1. However, inversions involving int22h‐2 are five times rarer than those involving its virtually identical copy: int22h‐3. Recent sequencing has indicated that int22h‐2 and int22h‐3 form the internal part of the arms of an imperfect palindrome so that int22h‐2, in the centromeric arm, has the same orientation as int22h‐1 and, upon recombination with int22h‐1, should produce deletions and duplications but not inversions. Aim: This work aims to provide rapid tests for all the mutations that can result from recombinations between the int22h sequences and to investigate whether int22h‐2‐related inversions causing hemophilia A arise in chromosomes, where the arms of the palindrome have recombined so that int22h‐2 and int22h‐3 swap places and orientation. Patients/methods: Twenty patients with int22h‐related inversions were examined together with a control and inversion carriers using reverse transcription‐polymerase chain reaction (RT‐PCR), long‐range PCR and sequencing. Results and conclusions: Analysis of mRNA in patients and a control provided evidence confirming the palindromic arrangement of int22h‐2 and int22h‐3 and the proposed inversion polymorphism that allows int22h‐2 to be in the telomeric arm of the palindrome and in opposite orientation to int22h‐1. New long‐range PCR reactions were used to develop a single tube test that detects and discriminates inversions involving int22h‐2 or int22h‐3 and a two‐tube test that can distinguish inversions, deletions, and duplications due to recombination between int22h sequences.

Mutation rates in humans. II. Sporadic mutation-specific rates and rate of detrimental human mutations inferred from hemophilia B

We estimated the rates per base per generation of specific types of mutations, using our direct estimate of the overall mutation rate for hemophilia B and information on the mutations present in the United Kingdoms population as well as those reported year by year in the hemophilia B world database. These rates are as follows: transitions at CpG sites 9.7x10-8, other transitions 7.3x10-9, transversions at CpG sites 5.4x10-9, other transversions 6.9x10-9, and small deletions/insertions causing frameshifts 3.2x10-10. By taking into account the ratio of male to female mutation rates, the above figures were converted into rates appropriate for autosomal DNA-namely, 1.3x10-7, 9.9x10-9, 7.3x10-9, 9.4x10-9, 6.5x10-10, where the latter is the rate for all small deletion/insertion events. Mutation rates were also independently estimated from the sequence divergence observed in randomly chosen sequences from the human and chimpanzee X and Y chromosomes. These estimates were highly compatible with those obtained from hemophilia B and showed higher mutation rates in the male, but they showed no evidence for a significant excess of transitions at CpG sites in the spectrum of Y-sequence divergence relative to that of X-chromosome divergence. Our data suggest an overall mutation rate of 2.14x10-8 per base per generation, or 128 mutations per human zygote. Since the effective target for hemophilia B mutations is only 1.05% of the factor IX gene, the rate of detrimental mutations, per human zygote, suggested by the hemophilia data is approximately 1.3.

The development and application of automated gridding for efficient screening of yeast and bacterial ordered libraries

An automated gridding procedure for the inoculation of yeast and bacterial clones in high-density arrays has been developed. A 96-pin inoculating tool compatible with the standard microtiter plate format and an eight-position tablet have been designed to fit the Biomek 1000 programmable robotic workstation (Beckman Instruments). The system is used to inoculate six copies of 80 x 120-mm filters representing a total of approximately 20,000 individual clones in approximately 3 h. High-density arrays of yeast artificial chromosome (YAC) and cosmid clones have been used for rapid large-scale hybridization screens of ordered libraries. In addition, an improved PCR library screening strategy has been developed using strips cut from the high-density arrays to prepare row and column DNA pools for PCR analysis. This strategy eliminates the final hybridization step and allows identification of a single clone by PCR in 2 days. The development of automated gridding technology will have a significant impact on the establishment of fully versatile screening of ordered library resources for genomic studies.

PRENATAL DIAGNOSIS OF XERODERMA PIGMENTOSUM: Report of the First Successful Case

Abstract Xeroderma pigmentosum (x.p.) was diagnosed prenatally by detecting, in amniotic cells cultured in vitro, the defect in the repair of ultraviolet-damaged D.N.A. which is pathognomonic of X.P. The procedure adopted is simple and reliable and can, therefore, be put to routine use for the prevention of X.P. The efficacy of preventive measures in X.P. is at least partly dependent upon the early postnatal detection of index cases as exemplified by the history of the family which is the subject of this report.

Detection of point mutations and a gross deletion in six Hunter Syndrome patients

We have used screening with the polymerase chain reaction and chemical mismatch detection of amplified cDNA to detect and characterize deletions and point mutations in six Hunter Syndrome patients. A high degree of mutational heterogeneity was observed. The first patient is completely deleted for the gene coding for alpha-L-iduronate sulfate sulfatase, while the second has a point mutation that creates a stop codon. The third patient shows a point mutation that creates a novel splice site that is preferentially utilized and results in partial loss of one exon in the RNA. Patients 4, 5, and 6 have point mutations resulting in single amino acid substitutions. Four of the six single-base changes observed in this study were examples of transitions of the highly mutable dinucleotide CpG to TpG. This study has demonstrated a procedure capable of detecting all types of mutation that affect the function of the IDS protein and should enable direct carrier and prenatal diagnosis for Hunter syndrome families.