Genetica

Pure & Appl. Chem., Vol. 63, No. 8, pp. 1089-1096, 1991 Printed in Great Britain. 0 1991 IUPAC

ADONIS

2069222091000965

Diagnosis of genetic diseases by DNA technology
Maurizio Ferrari, Laura Cremonesi, Paola Carrera and Pierangelo Bonini Istituto Scientific0 H.S.Raffaele, Lab. Centrale, Milano, Italy Abstract - The development of recombinant DNA technology has allowed the study of themolecular pathology of inherited diseases in man. Two main molecular approaches are employed: direct study for detection of molecular defects and indirect detection by linkage analysis. A variety of new technologies and their applications have provided a powerful new tool in the diagnosis of inherited diseases.

INTRODUCTION

During the last twenty years the recombinant DNA technology hasdeveloped very powerful and sensitive techniques, which are listed in Table 1, useful in the study and for the identification of the molecular defects of human inherited diseases. DNA technology has allowed an enormous increase of basic knowledge on inherited diseases and has had great effect on biodiagnostics; in general it has increased the knowledge on the human genome. The development of DNA baseddiagnostic tests for genetic disorders is still in progress. It will take time for experimental validation and for standardization of protocols. However their transferibility to the clinical field will depend on other factors, like the simplicity of procedures, the speed of execution and the cost of the analysis. Since 1 9 7 3 , 7 5 human genes have been mapped (ref. 1). Table 2 shows all thegenes mapped in the 1st edition of "Human Gene Mappingt'. Every year, during the Human Gene Mapping conference, the DNA Committee has three primary responsibilities: a) summarizing information on cloned and mapped human genes, b) summarizing information on polymorphisms detected by using molecular techniques, and c) the development of a nomenclature appropriate for loci identified by anonymous DNAprobes and assignment of symbols to those loci. Since 1 9 8 3 , these data have been collected also on Database to facilitate and to speed up the access (ref. 1). There are also others Database with the list of papers on cloned sequences both for genes and for anonimous sequences (ref. 2 ) . The number and chromosome distribution of cloned sequences and polymorphisms are shown in Fig. 1 and Table 3(10th edn of Human Gene Mapping). The nature of the genetic lesion has now been established at the DNA level in many diseases: the majority , 5 8 , are due to deletions, 5 2 are caused by point mutations and 3 4 by insertions, duplications and gene rearrangements (ref. 3 ) . A variety of chromosomal alterations have also been analysed and well characterized (ref. 4). The advent of molecular biologytechniques has expanded our ability to diagnose inherited diseases. In fact , it is possible to carry out the analysis at the DNA level and it is not necessary to know the gene product, which gives three advantages: a) the analysis should be done for example on limphocytes and not only on the disease target cells, b) it allows carrier detection and, c) it allows the molecular defectcharacterization.
Two

main molecular approaches are possible for genetic studies: a) indirect detection by linkage analysis, b) direct detection of the molecular alterations of genes.
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TABLE 1. Recombinant DNA technology

I
I

Probe
I

- Restriction
Endonucleases Cloning - Libraries - Electrophoresis - Blotting - Mapping - PFGE (a) - Chemical Cleavage

-Vectors Oligonucleotides P C R (b) Sequencing Probes DGGE ( c ) SSCP (d) RNAseA mismatch analysis

10 ktr

f
7kb

t A
- i

t
3kb

11

I

(a) Pulse Field Gel Electrophoresis (b) Polymerase Chain Reaction (c) Denaturing Gradient Gel Electrophoresis (d) Single Strand Conformational Polymorphism

?

B A:RwMctlm&teabstmt B : Eterrtrlctioa ate p w e n t

+ +

Fig. 2. RFLPs...