Diagnosis of human genetic disease using recombinant DNA

Recombinant DNA methodology has greatly increased our knowledge of the molecular pathology of the human genome at the same time as providing the means of diagnosing inherited disease at the DNA level. Direct detection and analysis of a wide range of genetic lesions are now possible using cloned gene or oligonucleotide probes or by direct sequencing of the disease gene(s). In addition, the use of restriction fragment length polymorphisms (RFLPs) within and around these genes as indirect genetic markers has potentiated the tracking of disease alleles in affected pedigrees in cases where direct analysis is not yet feasible. RFLPs associated with linked anonymous DNA segments may also be used not only to diagnose hitherto undetectable disease states, but also for the chromosomal localization of the loci responsible. We present here an update to our previous list of reports describing the direct and indirect analysis/diagnosis of human inherited disease. This compilation is intended to serve as a guide to current molecular genetic approaches in diagnostic medicine.     

Diagnosis of genetic disease using recombinant DNA. Supplement

Summary Recombinant DNA methodology has greatly increased our knowledge of the molecular pathology of the human genome at the same time as providing the means to diagnose inherited disease as the DNA level. We present here a list of recent reports of both direct and indirect analysis of human inherited disease which is intended to serve as a guide to current molecular genetic approaches to diagnostic medicine.     

Diagnosis of genetic disease using recombinant DNA

Summary Recombinant DNA technology promises to make an important contribution to the analysis and diagnosis of inherited human disease. Direct detection and analysis of various genetic defects at the DNA level are now possible using cloned gene or oligonucleotide probes. In addition, the use of restriction fragment length polymorphisms associated with linked DNA segments should permit not only the diagnosis of hitherto undetectable disease states but also the chromosomal localization of the loci responsible. The eventual isolation of disease loci should lead to a better understanding of the molecular basis of inherited disease.     

Diagnosis of genetic disease using recombinant DNA. Third edition

Summary Recombinant DNA methodology has greatly increased our knowledge of the molecular pathology of the human genome at the same time as providing the means to diagnose inherited disease at the DNA level. Direct detection and analysis of a range of genetic defects are now possible using cloned gene or oligonucleotide probes or by direct sequencing of the disease gene(s). In addition, the use of restriction fragment length polymorphisms (RFLPs) within and around these genes as indirect genetic markers has now potentiated the tracking of disease alleles in affected pedigrees in cases where direct analysis was not feasible. RFLPs associated with linked anonymous segments may also be used not only to diagnose hitherto undetectable disease states, but also for chromosomal localization of the loci responsible. We present here an updated list of reports describing both the direct and the indirect analysis/diagnosis of human inherited disease; it is intended to serve as a guide to current molecular genetic approaches in diagnostic medicine.Abbreviations ADG Annales de Genetique - AHG Ann. Hum. Genet. - AICHG Abstracts Int. Congress Hum. Genet. 7, Berlin, 1986 - AJH Am. J. Haematol. - AJHG Am. J. Hum. Genet. - AJMG Am. J. Med. Genet. - AN Aneuploidy - ANYAS Ann. New York Acad. Sci - APRT Adeninephosphoribosyltransferase - ASHG American Soc. Hum. Genet. Abstracts 34th Ann. Meeting - ATS VII Atherosclerosis VII, Eds. Fidge and Nestel, Elsevier - Arch. Neurol. Archieves of Neurology - Arch. Oph. Arch. Ophthalmol. - Atherosclr. Atherosclerosis - BBRC Biochim. Biophys. Res. Comm. - BJH Brit. J. Haematol. - BMJ Brit. Med. J. - BST Biochem. Soc. Transact. - CCG Cytogenet. Cell Genet. - CDC Carrier detection using clonality - CGC Cancer Genet. Cytogenet. - DEL Deletion - DETECT Mode of Detection - Dis. Marker Disease Markers - DUP Duplication - EJB Eur. J. Biochem. - EJI Eur. J. Immunol. - HGM8 Human Gene Mapping 8 (CCG, Vol. 40, 1–824, 1985) - HGM9 Human Gene Mapping 9 (CCG, Vol. 46, 1–824, 1987) - HGM10 Human Gene Mapping 10 (CCG, Vol. 51, 1–824, 1989) - HPRT Hypoxanthinephosphoibosyltransferase - HVR Hypervariable region - Hos. Prac. Hospital Practice - IMG Immunogenetics - INS Insertion - INV Inversion - IZ Inter-zeta - J. Mol. End. J. Molec. Endocrinol. - JAMA J. Amer. Med. Assoc. - JBC J. Biol. Chem. - JCB J. Cell. Biol. - JCEM J. Clin. Endocrinol. Metab. - JCI J. Clin. Invest. - JMD J. Inher. Metab. Dis. - JIMM J. Immunogenet. - JJCR Jpn. J. Cancer Res. - JJHG Jpn. J. Hum. Genet. - JMG J. Med.Genet. - JNR J. Neurosci. Res. - LH Loss of heterozygosity - MBM Mol. Biol. Med. - MCB Molec. Cell. Biol. - MCKUS McKusick catalogue number - MMP Mismatch pairing analysis - MODY Maturity onset diabetes of the young - MOL. END. Molec. Endocrinol. - MUT Mutation - NAR Nucl. Acids Res. - NEJM New Engl. J. Med. - Neurol. Sup. Neurology Supplement - OLIGO Detection of mutation by oligonucleotide hybridisation - OPG Ophthal. Pediatr. Genet. - PNAS Proc. Natl. Acad. Sci. USA - Ped. Res. Pediatric Res. - PM Point mutation - Pren. Diag. Prenatal Diagnosis - RE Restriction enzyme analysis - REAR Rearrangement - RFLP Indirect analysis using likned RFLP - SEQU Analysis by DNA sequencing - SCMG Somat. Cell Molec. Genet. - Thr. Res. Thrombosis Research - XIA X-inactivation analysis - ar alphoid repeat - atyp. atypical - breakp. breakpoint - def. deficiency - fruct. fructose - haem. haemoglobin - hered. hereditary - minisat. minisatellite - mt mitochondrial - neph. nephritis - persist. persistent - phosph. phosphorylase - resis. resistant - phosph. phosphorylase - resist. resistant - sev. several - synth. synthetase - var various     

Detection of DNA sequence polymorphisms by enzymatic amplification and direct genomic sequencing.

The discovery of RFLPs and their utilization as genetic markers has revolutionized research in human molecular genetics. However, only a fraction of the DNA sequence polymorphisms in the human genome affect the length of a restriction fragment and hence result in an RFLP. Polymorphisms that are not detected as RFLPs are typically passed over in the screening process though they represent a potentially important source of informative genetic markers. We have used a rapid method for the detection of naturally occurring DNA sequence variations that is based on enzymatic amplification and direct sequencing of genomic DNA. This approach can detect essentially all useful sequence variations within the region screened. We demonstrate the feasibility of the technique by applying it to the human retinoblastoma susceptibility locus. We screened 3,712 bp of genomic DNA from each of nine individuals and found four DNA sequence polymorphisms. At least one of these DNA sequence polymorphisms was informative in each of three families with hereditary retinoblastoma that were not informative with any of the known RFLPs at this locus. We believe that direct sequencing is a reasonable alternative to other methods of screening for DNA sequence polymorphisms and that it represents a step forward for obtaining informative markers at well-characterized loci that have been minimally informative in the past.     

Carrier detection in haemophilia B using two further intragenic restriction fragment length polymorphisms.

A normal human population has been screened for the existence of further restriction fragment length polymorphisms (RFLPs) in the clotting factor IX gene in addition to the TaqI polymorphism already characterised (1,2). Two polymorphic loci were found, both within 6 Kb of the TaqI polymorphism within the body of the factor IX gene. One of the polymorphisms has been shown to be due to either the presence or absence of a particular recognition site for the restriction enzyme XmnI. The other, visualised as a difference in fragment pattern produced by digestion with either HinfI or DdeI, has two allelic forms differing by a 50 bp element of inserted DNA. Sequence analysis has shown the inserted element to be in a region of Z type DNA sequence, the insertion representing a duplication of flanking sequence on either side. The two polymorphisms are inherited in simple Mendelian fashion and have both been used to diagnose haemophilia B carrier status. It is estimated that the combined use of these polymorphisms in the factor IX gene, despite linkage disequilibrium between the 3 polymorphic loci, should enable carrier status to be determined in approximately 66% of all haemophilia B families.     

Restriction fragment length polymorphisms associated with the gene for the major intrinsic protein of eye-lens fibre cell membranes in mice with hereditary cataracts.

Cloned cDNAs coding for eye-lens fibre cell-membrane proteins, MIP and MP70, were used to detect restriction fragment length polymorphisms (RFLPs) in genomic DNA from inbred mice with autosomally inherited cataracts. Whereas distinct RFLPs associated with the MIP gene were identified in the Cba Cat and Nct mutants, no such genetic variation was associated with the MP70 gene. RFLPs associated with the mouse MIP gene may provide informative DNA markers in gene linkage studies of murine hereditary cataracts.     

Restriction fragment length polymorphisms associated with the gene for the major intrinsic protein of eye-lens fibre cell membranes in mice with hereditary cataracts

Cloned cDNAs coding for eye-lens fibre cell-membrane proteins, MIP and MP70, were used to detect restriction fragment length polymorphisms (RFLPs) in genomic DNA from inbred mice with autosomally inherited cataracts. Whereas distinct RFLPs associated with the MIP gene were identified in the Cba Cat and Nct mutants, no such genetic variation was associated with the MP70 gene. RFLPs associated with the mouse MIP gene may provide informative DNA markers in gene linkage studies of murine hereditary cataracts.     

Genetic, cellular and immune approaches to disease therapy: past and future

Advances in immunology and molecular genetics have accelerated our understanding of the genetic and cellular basis of many diseases. At the same time, remarkable progress in recombinant DNA technology has enabled the development of molecular and cellular treatments for infectious diseases, inherited disorders and cancer. This Perspective is intended to give a sample of the progress over the past ten years in cellular, genetic and immune therapy of disease. During this time, monoclonal antibody technology and cellular transplantation have begun to come of age in biomedicine. Innovations in gene delivery have not only catalyzed the nascent field of human gene therapy, but may also ultimately impact human health by advancing recombinant vaccine technology.     

DNA restriction fragment length polymorphisms and heterozygosity in the human genome

Summary A list is presented of published reports of DNA polymorphisms found in the human genome by restriction enzyme analysis. While the list indicates the large number of restriction fragment length polymorphisms (RFLPs) detected to date, the information collated is insufficient to permit an estimate of heterozygosity for the genome as a whole. Data from our laboratory are therefore also presented on RFLPs detected using a random sample of cloned DNA segments. Such an analysis has permitted a first unbiassed estimate of heterozygosity for the human genome. Since this figure is an order of magnitude higher than previous estimates derived from protein data, the majority of polymorphic variation present in the human genome must, by implication, occur in noncoding sequences. In addition it was confirmed that enzymes containing the dinucleotide CpG in their recognition sequences detect more polymorphic variation than those that do not contain a CpG. Also presented are the clinical applications of DNA polymorphisms in the diagnosis of human genetic disease.Supported by the Deutsche Forschungsgemeinschaft     

Expression of tissue-specific genes in transgenic mice

The ability to introduce cloned genes into mouse germ line has been used for analyzing cis-acting DNA sequences involved in tissue-specific and developmental regulation of the introduced gene. Using this system we have attempted to produce a transgenic mouse model for human dominantly inherited disease, familial amyloidotic polyneuropathy. Recently the mutant transthyretin gene which is considered to be responsible for this disease has been cloned and well characterized at molecular level. We have produced transgenic mice by microinjecting human mutant gene. Amyloid deposition was observed in the mucosa of alimentary tract and renal glomeruli, suggesting that this approach is successful in establishing the mouse model for human genetic disease. In addition, these experiments suggest that the expression of the mutant gene is regulated normally during developmental process and that the cause of adult onset is not due to the dysregulation of this gene expression.     

Isolation of polymorphic DNA segments from human chromosome 21.

A somatic cell hybrid line containing only human chromosome 21 on a mouse background has been used as the source of DNA for construction of a recombinant phage library. Individual phages containing human inserts have been identified. Repeat-free human DNA subclones have been prepared and used to screen for restriction fragment length polymorphisms to provide genetic markers on chromosome 21. Nine independently isolated clones used as probes identified a total of 11 new RFLPs. Four of the DNA probes recovered from the library have been mapped unequivocally to chromosome 21 using a panel of somatic cell hybrid lines. A fifth probe detected an RFLP on chromosome 21 as well as sequences on other chromosomes. This set of RFLPs may now form the basis for construction of a genetic linkage map of human chromosome 21.     

Genetic Variants in Diseases of the Extrapyramidal System

Knowledge on the genetics of movement disorders has advanced significantly in recent years. It is now recognized that disorders of the basal ganglia have genetic basis and it is suggested that molecular genetic data will provide clues to the pathophysiology of normal and abnormal motor control. Progress in molecular genetic studies, leading to the detection of genetic mutations and loci, has contributed to the understanding of mechanisms of neurodegeneration and has helped clarify the pathogenesis of some neurodegenerative diseases. Molecular studies have also found application in the diagnosis of neurodegenerative diseases, increasing the range of genetic counseling and enabling a more accurate diagno-sis. It seems that understanding pathogenic processes and the significant role of genetics has led to many experiments that may in the future will result in more effective treatment of such diseases as Parkinson’s or Huntington’s. Currently used molecular diagnostics based on DNA analysis can identify 9 neurodegenerative diseases, including spinal cerebellar ataxia inherited in an autosomal dominant manner, dentate-rubro-pallido-luysian atrophy, Friedreich’s disease, ataxia with ocu-lomotorapraxia, Huntington''s disease, dystonia type 1, Wilson’s disease, and some cases of Parkinson''s disease.     

Molecular definition of red cell Rh haplotypes by tightly linked SphI RFLPs.

The Rh blood group system of human red cells contains five major antigens D, C/c, and E/e (the latter four designated "non-D") that are specified by eight gene complexes known as Rh haplotypes. In this paper, we report on the mapping of RH locus and identification of a set of SphI RFLPs that are tightly linked with the Rh structural genes. Using exon-specific probes, we have localized the SphI cleavage sites resulting in these DNA markers and derived a comprehensive map for the RH locus. It was found that the SphI fragments encompassing exons 4-7 of the Rh genes occur in four banding patterns or frameworks that correspond to the distribution and segregation of the common Rh haplotypes. This linkage disequilibrium allowed a genotype-phenotype correlation and direct determination of Rh zygosity related to the Rh-positive or Rh-negative status (D/D, D/d, and d/d). Studies on the occurrence of SphI RFLPs in a number of rare Rh variants indicated that Rh phenotypic diversity has taken place on different haplotype backgrounds and has arisen by diverse genetic mechanisms. The molecular definition of Rh haplotypes by SphI RFLP frameworks should provide a useful procedure for genetic counseling and prenatal assessment of Rh alloimmunization.     

Alphoid DNA polymorphisms for chromosome 21 can be distinguished from those of chromosome 13 using probes homologous to both.

Although alphoid DNA sequences shared among acrocentric chromosomes have been identified, no human chromosome 21-specific sequence has been isolated from the centromeric region. To identify alphoid DNA restriction fragment length polymorphisms (RFLPs) specific for chromosome 21, we hybridized human genomic DNA with alphoid DNA probes [L1.26; aRI(680),21-208] shared by chromosomes 13 and 21. We detected RFLPs with restriction enzymes ECoRI, HaeIII, MboI,StuI, and TaqI. The segregation of these RFLPs was analyzed in the 40 CEPH families. Linkage analysis between these RFLPs and loci previously mapped to either chromosome 13 or 21 revealed RFLPs that appear to be specific to chromosome 21. These polymorphisms may be useful as genetic markers of the centromeric region of chromosome 21. Different alphoid loci within the centromeric region of chromosome 13 were identified.     

A genetic map of mouse chromosome 1 near the Lsh-Ity-Bcg disease resistance locus

Isozyme and restriction fragment length polymorphism (RFLP) analyses of backcross progeny, recombinant inbred strains, and congenic strains of mice positioned eight genetic markers with respect to the Lsh-Ity-Bcg disease resistance locus. Allelic isoforms of Idh-1 and Pep-3 and RFLPs detected by Southern hybridization for Myl-1, Cryg, Vil, Achrg, bcl-2, and Ren-1,2, between BALB/cAnPt and DBA/2NPt mice, were utilized to examine the cosegregation of these markers with the Lsh-Ity-Bcg resistance phenotype in 103 backcross progeny. An additional 47 backcross progeny from a cross between C57BL/10ScSn and B10.L-Lshr/s mice were examined for the cosegregation of Myl-1 and Vil RFLPs with Lsh phenotypic differences. Similarly, BXD recombinant inbred strains were typed for RFLPs upon hybridization with Vil and Achrg. Recombination frequencies generated in the different test systems were statistically similar, and villin (Vil) was identified as the molecular marker closest (1.7 +/- 0.8 cM) to the Lsh-Ity-Bcg locus. Two other DNA sequences, nebulin (Neb) and an anonymous DNA fragment (D2S3), which map to a region of human chromosome 2q that is homologous to proximal mouse chromosome 1, were not closely linked to the Lsh-Ity-Bcg locus. This multipoint linkage analysis of chromosome 1 surrounding the Lsh-Ity-Bcg locus provides a basis for the eventual isolation of the disease gene.     

Introductory Lecture: Genetic Approaches to CNS Disorders with Particular

Abstract

The tools of molecular biology will bring the field of human genetics into a new era by permitting the analysis of the genetic contribution to disease. Most single gene disorders, inherited in a Mendelian fashion, will be molecularly diagnosed. In addition, the genetic susceptibility of common, complex diseases such a schizophrenia can be clarified, even though the conditions are not inherited as Mendelian characteristics. The mapping of the human genome will increase the rate at which new disease genes are identified and isolated. Finally, the development of genetically engineered animal models will help to dissect the steps involved in physiological and pathophysiological processes and thereby enhance our understanding of complex biological systems.     

RFLPs for Duchenne muscular dystrophy cDNA clones 9 and 10.

The complete 14-kb cDNA for the gene causing the X-linked recessive muscular dystrophy (MD) type Duchenne (DMD) and Becker (BMD) has recently been cloned and made available for deletion/duplication screening in patients. It detects 65 exon-containing nonpolymorphic HindIII fragments spread over a gene locus of about 2,000 kb. When the entire DMD cDNA is used, deletions/duplications can be found in about 65%-70% of affected patients, permitting direct carrier detection by densitometric scanning. But in cases where no deletion/duplication is detectable, RFLP analysis, specially favored within the gene, will be the method for carrier-status determination. Clones 9 and 10-1.2-kb and 0.7-kb fragments, respectively, of the 14-kb DMD cDNA--have been hybridized with human genomic DNA digested by nine different restriction enzymes. Five RFLPs, involving Asp700, PvuII, XbaI, and EcoRV sites, were detected, and Mendelian inheritance could be demonstrated. Since clones 9 and 10 are localized telomeric to the mutation-hot-spot region, their polymorphisms are thought to be very helpful as flanking markers for indirect carrier detection in families with a family history of DMD/BMD. Moreover, these RFLPs can be used for direct carrier detection or exclusion in families with patients showing a deletion/duplication in the region of p9 or p10.     

Linkage analysis of the D1 dopamine receptor gene and manic depression in six families.

Disturbances in dopaminergic activity may play an important role in the pathogenesis of manic depression. The effects of dopamine are mediated by at least five G protein coupled receptors, D1, D2, D3, D4 and D5. Recently, three separate research groups have cloned and characterized the D1 dopamine receptor, which localizes to 5q35.1. We undertook a linkage analysis between the D1 receptor polymorphisms and manic depression in six families in which segregation of the disease was consistent with autosomal dominant inheritance. A highly polymorphic flanking DNA marker, CRI-L1200, was also analyzed as the D1 gene RFLPs were relatively uninformative in our families. Multipoint analyses of manic depression and these DNA markers resulted in lod scores of less than -3.0 at the D1 locus, indicating that the D1 dopamine receptor gene does not confer an inherited susceptibility to manic-depressive illness in the families studied.