(8) is a diagrammatic representation of the staining pattern unique to ...
Transcript of (8) is a diagrammatic representation of the staining pattern unique to ...
THE HUMAN GENOME THROUGH THE EYES OF MERCATORAND VESALIUS
VICTOR A. McKUSICK
BALTIMORE
First, Mercator.Figure 1 provides a gene map of the human chromosomes. The banding
(8) is a diagrammatic representation of the staining pattern unique toeach chromosome. It bears no necessary relationship to the genes presentin particular parts of the chromosome. We estimate that the total numberof structural genes, that is, genes that determine the amino acid sequenceof proteins, is between 50,000 and 100,000 in man. Each gene locus issymbolized by a code most often of three letters. In all, over 375 gene locihave been, with various confidence (indicated by the letter style of thesymbol), assigned to a specific chromosome or in many instances aspecific region.
It is a matter of considerable intellectual satisfaction that we now know(15, 16, 17) that the gene for Rh blood type is on the short arm ofchromosome 1, that the gene for ABO blood type is on the end of thelong arm of chromosome 9, that the gene for insulin is on the short armof chromosome 11 (1), and that the genes for colorblindness and classichemophilia, two of the genes longest known in man, are in the unstainingor lightly staining region on the end of the long arm of the X chromosome.
Indeed, the first gene mapped to a specific chromosome in man or anymammal and perhaps in any organism was the colorblindness gene,assigned to the X chromosome by Professor E. B. Wilson at Columbia in1911. Wilson pointed out that the characteristic pedigree pattern ofcolorblindness, known from the work of Swiss ophthalmologist Homer inthe 1870's, could best be explained ifman has an XX, XY sex chromosomeconstitution and the colorblindness gene is on the X chromosome. Hal-dane showed that the colorblindness gene and hemophilia are closelylinked on the X chromosome, and Porter in our group in Baltimoreshowed that G6PD and colorblindness are closely linked. Boyer, also inBaltimore, showed that hemophilia and G6PD are closely linked. Thuswas defined a cluster of genes which subsequently by somatic cell hybrid-ization was found to be in the very terminal part of the long arm of theX chromosome. But I am getting ahead of my story.A considerable number of genes can be assigned to the X chromosome
Johns Hopkins University School of Medicine Physician-in-Chief, Johns Hopkins Hos-pital, Baltimore, MD 21205
66
THE HUMAN GENOME
by the method that Wilson used in 1911: characteristic pedigree pattern.That number of firmly assigned X-linked genes is presently about 115.Furthermore, by Mendelian pedigree pattern one can identify a consid-erable number of genes that are carried on one or another non-sexchromosome, or autosome. The catalogs of Mendelian traits in manwhich I have been maintaining for a long time provide that information.Table 1 indicates the numbers for both fully confirmed and tentativelyidentified autosomal phenotypes which, as far as we can tell, relate toseparate loci. The numbers have grown greatly since Verschuer's countin 1958 and through the five editions of Mendelian Inheritance in Man,1966 to 1978 (17). This reflects progress in genetic nosology, that is, theclassification and delineation of genetic diseases, as well as progress inidentification of genetic biochemical variation.
Progress is at least crudely indicated by the increasing size of the fivebooks. Incidently each is a different color. The fourth edition was blueand inevitably it was referred to by my students and residents as "bluegenes" and of course the last edition had to be "green genes." I supposeI am the only author that people ask, not "What is your next book goingto be on?", but, "What color is your next book going to be?" The grandtotal of autosomal and X-linked loci definitely or provisionally identifiedby the Mendelian approach and continuously updated, now over 3100, is,of course, still far short of the 50,000-100,000 figure. Per se the Mendelianapproach does not tell us which autosomal chromosome carries whichspecific gene. That information we now do have by other approaches forabout 275 genes, and this is information which has accumulated entirelyin the last 12 years.The first assignment of a specific gene to a specific autosome was
achieved in 1968 by one of my graduate students, Roger Donahue. Asevery good graduate student in human genetics should, Donahue studiedhis own chromosomes and found that he had one unusually long chro-mosome 1, with a peculiar uncoiled appearance of one segment of theproximal part of the long arm. By studying other members of his familyDonahue showed that the peculiar chromosome was a normal variation,a long chromosome 1 just as some people have a long nose. He showedthat it was transmitted as a dominant trait, present in many members ofhis family in heterozygous state. He then studied multiple markers, bloodgroups in particular, and found that a specific Duffy blood type tended tobe transmitted through the family in correlation with the anomalouschromosome 1. Thus the Duffy blood group locus, symbolized Fy in Fig.1, is situated on chromosome 1 in the vicinity of the centromere, aconclusion which has since been amply confirmed by other studies sincethat time.
67
VICTOR A. MCKUSICK
AI
1I 1., I
I.-I* *I
iI' __] @~
.4 E.~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
p
It
Ar
p~~~~
151 5 adawa a I TomI i if -,e A
0 x
14all'.
19 : 44 2a- ttU zz w
gal.. IfUPC, , )0.
PA. -.
tlb )I.
I I
Is
I be
aA
la
.1 -a
1tw~~~~~I Xe
C8
§C 2 il~il lit34ff i p
iB ...I.uhr F e rk'T
C'
IiiIL::::
.1
0)
!5
0.0
co
0
C;020)
r 140
a)
4-
bD
a
p
68
U.I%111-4,46mm
THE HUMAN GENOME
KEY TO GENE SYMBOLS SHOWN IN FIGURE 1 (with indication of chromosomaland in some instances regional location of the gene: e.g., 9q34 means band 34 of the longarm of chromosome 9, 2p23 means band 23 of the short arm of chromosome 2)
ABO = ABO blood group - 9q34ACEE = Acetylcholinesterase expression - chr. 2ACON-M = Aconitase, mitochondrial - 22qll-qterACON-S = Aconitase, soluble - 9pter-p13ACP1 = Acid phosphatase-i - 2p23ACP2 = Acid phosphatase-2 - 11p12-cenACY1 = Aminoacylase-l - 3pter-q13ADA = Adenosine deaminase - 20ql3-qterADCP1 = Adenosine deaminase complexing protein-i - chr. 6ADCP2 = Adenosine deaminase complexing protein-2 - chr. 2ADK = Adenosine kinase - 10qll-q24AH3 = Adrenal hyperplasia III (21-hydroxylase deficiency) (6p2105-6p23)AHH = Aryl hydrocarbon hydroxylase - 2pAK1 = Adenylate kinase-i (soluble) - 9q34AK2 = Adenylate kinase-2 (mitochondrial) - lp32-p34AK3 = Adenylate kinase-3 (mitochondrial) - 9pter-pl3AL = Lethal antigen: 3 loci - al,a3 on llpl3-pter;a2 on llql3-qterAlb = Albumin - 4qll-ql3AMY1 = Amylase, salivary - lplAMY2 = Amylase, pancreatic - lplAnl = Aniridia, type 1 (chr.2; linked to ACP1)APRT = Adenine phosphoribosyltransferase - 16qARS-A = Arylsulfatase A - chr. 22ARS-B = Arylsulfatase B - chr. 5ASD2 = Atrial septal defect, secundum type (chr. 6; linked to HLA)ASH = Asymmetric septal hypertrophy (chr. 6; linked to HLA)ASL = Argininosuccinate lyase - 7pter-q22ASS = Argininosuccinate synthestase - chr. 9AT3 = Antithrombin III (chr. 1)AV12M1 = Adenovirus-12 chromosome modification site-i - lq42-43AV12M2 = Adenovirus-12 chromosome modification site-2 - lp36AV12M3 = Adenovirus-12 chromosome modification site-3 - iq21AV12-17 = Adenovirus-12 chromosome modification site-17 - 17q21-q22AVP = Antiviral protein - 21q21-qterAVr = Antiviral state regulator - chr. 5
,B2M (B2M) = Beta-2-microglobulin - 15ql4-q21BCT-1 = Branched chain amino acid transferase-1 - chr. 2BCT-2 = Branched chain amino acid transferase-2 - chr. 19BEVI = Baboon M7 virus infection - chr. 6BF = Properdin factor B - chr. 6 (in MHC)BVIN = BALB virus induction, N-tropic - chr. 15BVIX = BALB virus induction, xenotropic - chr. 11
C2 = Complement component-2 - chr. 6 (in MHC)C4F = Complement component-4 fast - chr. 6 (in MHC)C4S = Complement component-4 slow - chr. 6 (in MHC)
69
VICTOR A. MCKUSICK
CaeCATCBCB3SCF7ECGChCHOLCKBBCMLCoCOilC012C031CSCSMT (or CSH)
DCEDHPRDia-lDIA-4DMJDNCDNCMDoDTS
ElE2EllSEBS1EBVEGFREllEMP130EMP195ENO1ENO2Es-ActEsA4EsD
FGPSFGRATFHFNFS
aFUC (FUCA)FUSEFy
= Cataract, zonular pulverulent (chr. 1; linked to Fy)= Catalase - llp= Colorblindness (deutan and protan) (Xq26-Xqter)= Coxsackie B3 virus susceptibility - chr. 19= Clotting factor VII expression (chr. 8)= Chorionic gonadotropin (chr. 10 and 18; chr. 5 or 6)= Chido blood group - same as C4S= Hereditary hypercholesterolemia - chr. 6 (?linked to HLA)= Creatine kinase, brain type - chr. 14= Chronic myeloid leukemia - 22ql2= Colton blood group (chr. 7)= Collagen I alpha-i chain - chr. 7 and 17= Collagen I alpha-2 chain - chr. 7 and 17= Collagen III alpha-1 chain - chr. 7= Citrate synthase, mitochondrial - chr. 12= Chorionic somatomammotropin - (chr. 17)
= Desmosterol-to-cholesterol enzyme - chr. 20= Quinoid dihydropteridine reductase - chr. 4= NADH-diaphorase - chr. 22= Diaphorase-4 - chr. 16= Juvenile diabetes mellitus (chr. 6; ?linked to HLA)= Lysosomal DNA-ase - chr. 19= Cytoplasmic membrane DNA - 9qh= Dombrock blood group (?chr. 1 or 4)= Diphtheria toxin sensitivity - 5ql5-qter
= Pseudocholinesterase-l - (?chr. 3; linked to TfO= Pseudocholinesterase-2 - 16cen-q22- Echo 11 sensitivity - 19q= Epidermolysis bullosa, Ogna type (chr. 10)= Epstein-Barr virus integration site , chr. 14= Epidermal growth factor, receptor for - chr. 7= Elliptocytosis-1 - (lp; linked to Rh)= External membrane protein-130 - chr. 10= External membrane protein-195 - chr, 14= Enolase-l - lp36-lpter= Enolase-2 - chr. 12= Esterase activator - chr. 4 or 5= Esterase-A4 - llcen-q22= Esterase D - 13ql4
= Folylpolyglutamate synthetase - chr. 9= Formylglycinamide ribotide amidotransferase - chr. 4 or 5= Fumarate hydratase - lq42-qter= Fibronectin - chr. 8, 11= Fragile site, observed in cultured cells, with or without folate deficient
medium, or BrdU - 2qll; 9q33; 10q23; 10q25; llql3; l6pl24; 16q22;20pll; Xq27
= Alpha-L-fucosidase - lp32-p34= Polykaryocytosis inducer - chr. 10= Duffy blood group - lql3
70
THE HUMAN GENOME
Gal+-ActaGALAaGALB,BGAL-1,BGAL-2GALEGALKGALTGAPDGARSGCGDHGHGHLaGLU (GLUA)GLUCGLO1GOT-MGOT-SG6PDGP130GPIGPT1GPx1GSRGSSGmGUKI & 2GUS
H4HADHHaFHbaHbBHbSHbyA,GHbyrHbeHb;HchHEM-AHexAHexBHGPRTHHPFH
HK1HLA(A-D)HLA-DRHpaHpaI
= Galactose + activator - chr. 2= Alpha-galactosidase A (Fabry disease) - Xq22-q24= Alpha-galactosidase B - 22ql3-qter= Beta-galactosidase-1 - 3pter-q13= Beta-galactosidase-2 - 22ql3-qter= Galactose-4-epimerase - 1p21-pter= Galactokinase - 17q21-q22= Galactose-l-phosphate uridyltransferase - 9pl3 or 9p22= Glyceraldehyde-3-phosphate dehydrogenase - 12p122-pter= Glycinamide ribonucleotide synthetase - chr. 21= Group-specific component - 4q11-q13= Glucose dehydrogenase - 1p21-pter (1p32-pter)= Growth hormone - chr. 17= Growth hormone like - chr. 17= Alpha-glucosidase - chr. 17= Neutral alpha-glucosidase C - chr. 15= Glyoxylase I - 6p21-6p22= Glutamate oxaloacetate transaminase, mitochondrial - chr. 16= Glutamate oxaloacetate transaminase, soluble - 10q24-q26= Glucose-6-phosphate dehydrogenase - Xq25-qter= Granulocyte glycoprotein - 7q22-qter= Glucosephosphate isomerase - chr. 19= Glutamate pyruvate transaminase, soluble - chr. 10= Glutathione peroxidase-1 - 3pl3-ql2= Glutathione reductase - 8p2l= Glutamate-gamma-semialdehyde synthetase - chr. 10= Immunoglobulin heavy chain - chr. 6,7,8 (see Igh)= Guanylate kinase-1 & 2 - lq32-q42= Beta-glucuronidase - chr. 7
= Histone H4 and 4 other histone genes - chr. 7= Hydroxyacyl-CoA dehydrogenase - chr. 7= Hageman factor - 7q35= Hemoglobin alpha chain - chr. 16= Hemoglobin beta chain - 11p1205-p1208= Hemoglobin delta chain - llpl205-p1208= Hemoglobin gamma chains, ala or gly as AA 136 - 11p1205-p1208= Hemoglobin gamma regulator - 11p1205-p1208= Hemoglobin epsilon chain - 11p1205-p1208= Hemoglobin zeta chain - chr. 16= Hemochromatosis (chr. 6; linked to HLA)= Classic hemophilia - Xq26-Xqter= Hexosaminidase A - 15q22-15qter= Hexosaminidase B - 5cen-ql3= Hypoxanthine-guanine phosphoribosyltransferase - Xq26-qter= Heterocellular hereditary persistence of fetal hemoglobin - 1lp1205-
p1208= Hexokinase-1 - 10pter-q24= Human leukocyte antigens - 6p2l05-p23= Human leukocyte antigen, D-related - 6p2l05-p23= Haptoglobin, alpha - chr. 16= Hpa I restriction endonuclease polymorphism - llpl205-pl208
71
VICTOR A. MCKUSICK
HVS = Herpes virus sensitivity (chr. 3 and 11)H-Y = Y histocompatibility antigen (Y chr.)
IDH-M = Isocitrate dehydrogenase, mitochondrial - 15q21-qterIDH-S = Isocitrate dehydrogenase, soluble - 2q11 or 2q32-qterIfl = Interferon-1 - 2p23-qterIf2 = Interferon-2 (chr. 5)If3 = Interferon-3 (chr. 9)IgAS = Immunoglobulin heavy chains attachment site - chr. 2Igh = Immunoglobulin heavy chains (mu, gamma, alpha) - chr. 14 (see Gm)Ins = Insulin - chr. 11ITP = Inosine triphosphatase - 20p
Jk = Kidd blood group - 7q
Km = Kappa immunoglobulin light chains, Inv (chr. 7)
LAP = Laryngeal adductor paralysis - (chr. 6; linked to HLA)LCAT = Lecithin-cholesterol acyltransferase - (16q22; linked to Hp alpha)LDH-A = Lactate dehydrogenase A - llpl203-pl208LDH-B = Lactate dehydrogenase B - 12pl21-pl22LDH-C = Lactate dehydrogenase C - (12p; linked to LDH-B in pigeon)LIPA = Lysosomal acid lipase-A - chr. 10Lp = Lipoprotein - Lp - chr. 13LTRS = Leucyl-tRNA synthetase - chr. 5
,B2M (B2M) = Beta-2-microglobulin - 15q22-15qter (15ql2-q21)M7VS1 = Baboon M7 virus sensitivity-i - chr. 19aMAN-A = Cytoplasmic alpha-D-mannosidase - 15qll-qteraMAN-B = Lysosomal alpha-D-mannosidase - l9pter-q13MDH-M = Malate dehydrogenase, mitochondrial - 7p22-q22MDH-S = Malate dehydrogenase, soluble - 2p23ME1 = Malic enzyme, soluble - 6p21-ql6MHC = Major histocompatibility complex - 6p2105-p23MLC-W = Mixed lymphocyte culture, weak (chr. 6)MNSs = MNSs blood group - 4qMPI = Mannosephosphate isomerase - 15q22-qterMRBC = Monkey red blood cell receptor - chr. 6MTR = 5-Methyltetrahydrofolate: L-homocysteine S-methyltransferase, or
tetrahydropteroyl-glutamate methyltransferase - chr. 1
NAG = Non-alpha globin region - lipl205-1208NDF = Neutrophil differentiation factor (chr. 6)NP = Nucleoside phosphorylase - 14q13NPa = Nail-patella syndrome - (9q3; linked to ABO)
OPCA1 = Olivopontocerebellar atrophy I - (chr. 6; linked to HLA)
P = P blood group (chr. 6)PA = Plasminogen activator (chr. 6)PDB = Paget disease of bone - (chr. 6; ?linked to HLA)PepA = Peptidase A - 18q23-18qter
72
THE HUMAN GENOME
PepB = Peptidase B - 12q21PepC = Peptidase C - 1q25, or 1q42PepD = Peptidase D (chr. 19)PepS = Peptidase S - 4pter-q12PFK-F = Phosphofructokinase, fibroblast - chr. 106PGD = 6-Phosphogluconate dehydrogenase - 1p34-pterPGK = Phosphoglycerate kinase - Xql3PGM1 = Phosphoglucomutase-1 - lp32; lp221-p311; 1p33-p34PGM2 = Phosphoglucomutase-2 - 4pl4-ql2PGM3 = Phosphoglucomutase-3 - 6qPGP = Phosphoglycolate phosphatase - 16pPK3 = Pyruvate kinase-3 - 15ql4-qterPKU = Phenylketonuria (ip; linked to AMY)PL = Prolactin - chr. 6PP = Inorganic pyrophosphatase - 10pter-q24PRPPAT = Phosphoribosylpyrophosphate amidotransferase - 4pter-q21PRPPS = Phosphoribosylpyrophosphate synthetase - X chr.PRAIS = Phosphoribosylaminoimidazole synthetase - chr. 21PVS = Polio virus sensitivity - 19qPWS = Prader-Willi syndrome - 15q11-q12
RB1 = Retinoblastoma-1 - 13ql2-ql4; 13q21-22rC3b = Receptor for C3b - chr. 6 (in MHC)rC3d = Receptor for C3d - chr. 6 (in MHC)Rg = Rodgers blood group - same as C4FRh = Rhesus blood group (1p32-pter)RN5S = 5S RNA gene(s) - lq42-q43RP1 = Retinitis pigmentosa-1 (chr. 1)rRNA = Ribosomal RNA - 13pl2, 14pl2, 15pl2, 21pl2, 22p12RwS = Ragweed sensitivity - (chr. 6; ?linked to HLA)
SA6 = Surface antigen 6 - chr. 6SA7 = Surface antigen 7 - 7p12-pterSAil = Surface antigen 11 - lipSA12 = Surface antigen 12 - chr. 12SA17 = Surface antigen 17 - chr. 17SA21 = Surface antigen 21 - chr. 21Sc = Scianna blood group - (lp32-p34)Sf = Stoltzfus blood group - (4q; linked to MNSs)SHMT = Serine hydroxymethyltransferase - chr. 12SOD1 = Superoxide dismutase, soluble - 21q11SOD2 = Superoxide dismutase, mitochondrial - 6q21SORD = Sorbitol dehydrogenase - 15pter-q21Sph 1 = Spherocytosis, Denver type (8pll or chr. 12)SS = Steroid sulfatase (?Xp22-pter)
TC2 = Transcobalamin II - (9q; ?linked to ABO)TDF = Testis determining factor - prob. same as H-YTf = Transferrin - ?chr. 3TK-M = Thymidine kinase, mitochondrial - chr. 16TK-S = Thymidine kinase, soluble - 17q21-q22TPI-1 & 2 = Triosephosphate isomerase-i & 2 - TPI-1 on 12pl2.2-pter
73
VICTOR A. MCKUSICK
tsAF8 = Temperature-sensitive (AF8) complement - chr. 3Tyr = Tyrosinase - (?llp)Tys = Sclerotylosis - (4q; linked to MNSs)
UGPP1 = Uridyl diphosphate glucose pyrophosphorylase-1 - lq21-q23UGPP2 = Uridyl diphosphate glucose pyrophosphorylase-2 - chr. 2UMPK = Uridine monophosphate kinase - lp32UP = Uridine phosphorylase - chr. 7UPS = Uroporphyrinogen I synthase - chr. 11
WAGR = Wilms tumor - aniridia/ambiguous genitalia/mental retardation -
llpl3
WTRS = Tryptophanyl-tRNA synthetase - chr. 14WS1 = Waardenburg syndrome-1 - (chr. 9; ?linked to ABO)Xg = Xg blood group (X chr., ?Xp2)
This was 1968 and at about the same time the method of somatic cellhybridization for chromosomal assignment of genes was developed byWeiss and Green with thymidine kinase as the first assignment, tochromosome 17.
In the method of somatic cell hybridization cells from two differentspecies, for example, a human cell and a mouse cell are fused. The nucleusof the hybrid cell becomes one, the chromosomes are intermixed and thecell expresses properties of both the mouse and man. The clones derivedfrom the dividing hybrid cell have the useful property that whereas theyretain a full complement of mouse chromosomes, they lose, largely atrandom, the human chromosomes so that one ends up with subcloneshaving various combinations of human chromosomes, perhaps only onehuman chromosome. If one has a method of selection one can assumethat there is only one particular human chromosome. That was done inthe classic experiment of Weiss and Green, fusing a mouse cell that isdeficient in thymidine kinase with a human cell and applying selectivefactors in the medium such that the hybrid cell would not survive unlessthe human chromosome carrying thymidine kinase were present. Thatselective medium is called HAT and was introduced into human somaticcell genetics by John W. Littlefield, our Professor of Pediatrics at Hop-kins. Thus, human chromosome 17 carries the thymidine kinase locus,specifically the cytoplasmic or soluble form of thymidine kinase. (Thereis a genetically separate form of thymidine kinase which in the mitochon-drion and is determined by a gene on chromosome 16.)
Parenthetically, by the methods of molecular genetics we know nowthat chromosome 17 also carries the gene for growth hormone and thegene for placental lactogen (called here chorionic somatomammotropin,CSMT) and probably genes for collagen.
74
-4 --e
bb
00 t LO - u
+ +++t~- ul~ r-4 CO
LO LOC '-t
.8
4
i bL
+++ +
-4 +J
S CO OO B
'-4c
0~X0 00 tS
o~~ow co w
COb t-
Ca i :eqLOCq c
4))
4J--HIV
75
VICTOR A. MCKUSICK
TABLE MBNumber ofLoci (From Mendelian Inheritance in Man, Aug 1980)
Established Tentative Total
Autosomal 1411 1485 2896X-linked 115 110 225Total 1526 1595 3121
Note that in the method of somatic cell hybridization interspeciesdifferences in particular proteins substitute for interallelic differenceswhich are the basis of mendelian genetic analysis and that the randomloss of human chromosomes from the mouse-man hybrid simulates seg-regation and assortment that occurs in meiosis. Thus, Haldane referredto this approach as an "alternative to sex" and Pontecorvo called thisand other approaches "parasexual" methods.We have had a series of five international workshops on human gene
mapping, the first in 1973, the most recent in 1979 in Edinburgh.* Table2 indicates the progress in mapping of the autosomes in these conferences.In Table 3, I have broken down the assignments according to the methodused. Almost a fourth of them have been made by the family method.About 60% of them have been made by the method of somatic cellhybridization. About 6% have been made independently by both of thesemethods and the rest have been made by other methods. In recent timesthe methods of molecular genetics have been particularly productive. Bythe techniques of recombinant DNA one can clone, purify, and amplifyspecific human genes or short chromosomal segments (14). One can usethese (or the complementary DNA (cDNA) synthesized with transversetranscriptase from the messenger RNA for specific proteins) as probes,hybridizing them in a radioactive form to the DNA and thereby deter-mining what chromosome they home in on. One can combine thisapproach with somatic cell hybridization, for example, or with chromo-somes that are sorted out with the fluorescence-activated cell sorter thathas been so useful in other applications.
Restriction enzymes have proved a valuable scalpel for dissecting the
* The Proceedings of the five workshops have been published in the March of DimesBirth Defects: Original Article Series and in Cytogenetics and Cell Genetics, as follows:
BD:OAS Cytogenet Cell GenetHGM-1 X(3): 1-216, 1974 13: 1-216, 1974HGM-2 XI(3): 1-310, 1975 14: 162-480,1975HGM-3 XII(7): 1-452, 1975 16: 1-462, 1976HGM-4 XIV(4): 1-730,1978 22: 1-730, 1978HGM-5 XV(11): 1-236, 1979 25: 2-236, 1979
76
THE HUMAN GENOME
TABLE IINumber of loci assigned at each of the five human gene mapping workshops
Number of autosomal assignments* X-chromosome assign-mentsConference P Inconsist-
ProviM ent (incl. Confirmed Total Confirmed In limbo
New Haven (1973) 28 5 31 64 88 67Rotterdam (1974) 32 6 48 86 91 70Baltimore (1975) 46 7 72 125 95 80Winnipeg (1977) 82 11 83 176 102 96Edinburgh (1979) 87 20 123 230 112 101
* Numbers of autosomal assignments for first four conferences provided by Donald andHamerton: Cytogenet Cell Genet 22: 5, 1978.
TABLE IIIMethod ofAutosomal Assignment (Dec 1979)
By family study 58By somatic cell hybridization 139Independently by both of above methods 14By other methods 32
Total 242
human genome. These are endonucleases that are derived from variousbacteria and cleave the DNA at various sites depending on the nucleotidespresent. The pattern of fragments produced by restriction enzyme diges-tion of specific fragments of DNA gives one insight into the structure ofthat segment. These patterns are called Southern blots. The restrictionenzymes are to the field ofDNA structure what trypsin or chymotrypsinwere to the study of protein structure 20 and 25 years ago, and theSouthern blot is analogous to Vernon Ingram's fingerprints of hemoglo-bin, for example.
Fine structure mapping of chromosomes by the approaches of molec-ular genetics is no better illustrated than by that of a part of the shortarm of 11 that carries the gene for the beta hemoglobin chain and relatedgenes: the gene for the epsilon chain of embryonic hemoglobin, the genesfor the gamma chains of fetal hemoglobin and the genes for the delta andbeta chains of hemoglobin A2 and hemoglobin A, respectively (Fig. 2).The same approach reveals the internal structure of the gene, with somesurprises: The coding parts of the beta hemoglobin gene is not continuousbut is in three segments interrupted by two intervening sequences, thefunction of which is still not thoroughly understood (Fig. 3).Except for the passing reference to the scalpel, I have used to this point
a cartographic mataphor, speaking of mapping the chromosomes in man.The landmarks in the maps are the bands revealed by special staining.
77
VICTOR A. MCKUSICK
kiinbases (kb)
60 50 40 30 20 10 0I II I1I11I11I1II1II1I11I1 IIIIIIIII I I I II111 I I
*02 e G-y Aty 01 6 _
Order of activation in ontogenyDirection of transcription -
3@2 t1 Oal a2 al
FIG. 2. Map of the beta-globin (of hemoglobin) region of the short arm of chromosome11 (above) and the alpha-globin (of hemoglobin) region of chromosome 16. Map distance isindicated in thousands of nucleotide basepairs (kilobases, kb). After Proudfoot NJ, ShanderMHM, Manley JL, Gefter ML, Maniatis T: Structure and in vitro transcription of humanglobin genes. Science 209: 1329-1336, 1980.
0 200 400 600 800 1000 1200 1400 1600
a l31 32 99 100 141
30 31 104 105 146
FIG. 3. Map of the alpha and beta globin genes. Map distance is indicated by number ofnucleotide basepairs (bp). From Proudfoot et al, loc cit.
The unitage of distance in the maps is recombination fraction, observedin family studies, or the centimorgan (as in Fig. 4), a derivative unitagebased on recombination fraction. In recent times the unitage has beennumber of base pairs (e.g., kilobases); see (Figs. 2 and 3).This is the cartographic metaphor, but an anatomic metaphor is equally
apt. The chromosomes and the linear arrangement of the genes theycarry are parts of human anatomy. One can look at the human genomeequally appropriately through the eyes of Mercator or through the eyesof Vesalius.
I am struck by the parallelism in the careers of Mercator, the geogra-pher-cartographer (Fig. 5A), and Vesalius (Fig. 5B), the physician-ana-tomist. Both were born in present day Belgium, in 1512 and 1514 respec-tively. They worked for many years within 20 miles of each other,Vesalius in Brussels, Mercator in Louvain. Mercator and Vesalius bothchallenged ancient authority, Ptolemy and Galen, respectively. Each inthe title of his magnum opus referred to his object of study as "Fabrica":Mercator spoke of Fabrica mundi (Fig. 6A); of course, Vesalius's greatwork was entitled "De Corporis Humanis Fabrica" (Fig. 6B).Now, Vesalius.Particularly in analyzing the significance of the mapping information
that has accumulated to date, it is useful to think in an anatomic frame.
78
THE HUMAN GENOME
F C2no recombination C4with HLA-B [Bf
(R) AH3 B C AHCH6q centromere
- 0.8 cM 0.8 cM-0.2 cM
I l! l-15cM - 3cM
l l
on 6p2105-p2300FIG. 4. Map of the part of chromosome 6 that includes the major histocompatibility
complex (MHC). A, B, C, D(R) = HLA-A, HLA-B, HLA-C, HLA-D(R). AH3 = adrenalhyperplasia III, or 21-hydroxylase deficiency. HCH = hemochromatosis. For other locussymbols, see key for Fig. 1. Map distances are in centimorgans (cM).
Let us look at the morbid anatomy, the comparative anatomy andevolution, the functional anatomy, the developmental anatomy, and eventhe applied anatomy of the human genome.As indicated in Table 4, for an ever increasing number of diseases the
chromosomal location of the mutant gene responsible is known. In manyof these instances this location is known because the enzyme which isdeficient has been assigned to a specific location. In most of thesedisorders the evidence is strong that it is indeed the structural gene forthe enzyme that is mutant in the given disease.Some of these assignments are at the borderline between chromosomal
aberrations and gene mutations as we usually view them. For example, asmall deletion in the short arm of chromosome 11, which was totallyundetectable before chromosome banding, leads to a syndrome of Wilmstumor, aniridia, gonadoblastoma and some other abnormalities (7). Asmall deletion on the long arm of chromosome 13 leads to retinoblastoma(25). A specific abnormality of chromosome 22 ("the Philadelphia chro-mosome") leads to chronic myeloid leukemia (21). A variety of abnor-malities involving the proximal part of the long arm of chromosome 15leads to the Prader-Willi syndrome (27). This syndrome, which in thepast would perhaps have been called Froelich syndrome, at least when itoccurred in males, is characterized at birth by hypotonia and feedingproblems and later by extreme obesity with paradoxically small handsand feet and with variable mental retardation. Like other problemslumped together as eating disorders, this has often been viewed as apsychiatric state and the organic basis revealed by the chromosomalaberration has been in my experience a relief to the families of the
79
VICTOR A. MCKUSICK
i00'..!£
Ie i
GE1EAPD MIRCAr(lPJS RVFLLMVNDA.NI !FS2MiiC
PWVCRYM ET SEX -_ ACI WrASVI ER A PSVM STYDII fCAV.YA DZIPINGI CVIkA8&ATr FANC. NOG. Cy). ), LXXIV.He d - iiaim---
* A, S of _ _ *2_i u He A, Y~~sk4.,-- = AFIG. 5. A. Gerard Mercator (1512-1594), geographer-cartographer. From Tooley (22). B.
Andreas Vesalius (1514-1564), physician-anatomist. From Mumey (19).
80
THE HUMAN GENOME
I1iiJ
81
82 VICTOR A. MCKUSICK
6ubs .WU'fnie,Afr#or,Afixr LiAmeriexltuA-dlrigtqtauX~ris Ss~xidz .f4t'Iseam daxe._.ttafvan'rtfJdu~drEms *rt ef r.m'udm ".
FIG. 6. A. Title-page of Mercator's Atlas (Mercator was the first to use the term atlasfor a collection of maps.) From Tooley (23). B. Title-page of Vesalius's Fabrica. FromMumey (19).
..
AW
-rd I W uY4776
4 C, jvmk..AND6 N
:;OM!.,It
.A. k Ak&Arl
'- / f. `- 'Ir
THE HUMAN GENOME 83
ANDREAE VESALIEBR V XE L L F N St S, S CHOLAE
mcdicorum iatauinacprofefforis~deHumanicorporisfabrics
Lbrit prn.
.
VICTOR A. MCKUSICK
TABLE IVGenetic Diseases for Which the Mutation Has Been Mapped to a Specific Autosome
A. DISORDERS OF CARBOHYDRATE METABOLISMFucosidosis 1p*Galactosemia 9pGalactokinase deficiency 17qGalactose epimerase deficiency lpGlycogen storage disease II 17
B. DISORDERS OF AMINO ACID METABOLISMClassic phenylketonuria 1(?)Atypical phenylketonuria 4
C. DISEASES OF LIPID METABOLISMNorum disease (LCAT deficiency) 16q
D. LYSOSOMAL STORAGE DISEASES(Fabry disease Xq)Generalized gangliosidosis 3Glycogen storage disease II 17Lysosomal acid phosphatase deficiency lipMannosidosis 19Metachromatic leukodystrophy 22q(MPS II (Hunter syndrome) X)MPS VI (Maroteaux-Lamy syndrome) 5MPS VII (Sly syndrome) 7Sandhoff disease 5qTay-Sachs disease 15qWolman disease 10
E. UREA CYCLE DISORDERSArgininosuccinicaciduria 7Citrullinemia 9(Ornithine transcarbamylase deficiency X)
F. CONGENITAL NONSPHEROCYTIC HEMOLYTIC ANEMIAGlucosephosphate isomerase deficiency 19Hexokinase deficiency 10Triosephosphate isomerase deficiency 12p
G. OTHER HEMATOLOGIC DISORDERSElliptocytosis lpSickle cell anemia lipThalassemias lip, 16p
H. IMMUNE DEFICIENCY DISEASES**Adenosine deaminase deficiency 20qNucleoside phosphorylase deficiency 14qC2 deficiency 6pC4 deficiency 6pTranscobalamin II deficiency 9q
* The numbers refer to chromosome carrying the particular locus, with arm when known.p = short arm; q = long arm. Three X-linked disorders are listed for sake of completenessin certain categories. The list is not exhaustive.
* * At least six X-linked immunodeficiencies are known (Lederman HM, Mak H, PeppleJM, Winkelstein JA: X-linked immunodeficiency diseases. Johns Hopkins Med J 147: 33-39, 1980).
84
THE HUMAN GENOME
TABLE IV (continued)
I. ENDOCRINOPATHIESCongenital adrenal hyperplasia (21-hydroxylase deficiency) 6pIsolated growth hormone deficiency 17Hyperproinsulinemia lip
J. MALIGNANT NEOPLASMWilms tumor (WAGR syndrome) lipRetinoblastoma 13qChronic myeloid leukemia 22q
K. MISCELLANEOUSAcatalasemia lipAcute intermittent porphyria 11Analbuminemia 4qCataract, zonular pulverlent 1Hemochromatosis 6pNail-patella syndrome 9qOlivopontocerebellar atrophy I 6Prader-Willi syndrome 15qTetrahydrofolate methyltransferase deficiency 1
afflicted. This finding adds another stone to the foundations of an organicbasis of morbid obesity.
Fine structure analysis by the methods of molecular genetics revealsvarious types of morbid anatomy: in the thalassemias, for example, andin the several forms of hereditary persistence of fetal hemoglobin. Inbeta-zero-thalassemia, the methods reveal point mutation in the seven-teenth codon of the beta gene, the change being from lysine to "stop" sothat no beta chain is synthesized. The methods show various extents ofdeletion of beta and the neighboring delta gene in others of the thalas-semias as well as various deletions of alpha-globin genes in the alpha-thalassemias. The methods also disclose deletion of a noncoding regionbetween the gamma globin genes and the delta gene, a region importantapparently in the switch from fetal hemoglobin to adult hemoglobin, inhereditary persistence of fetal hemoglobin.Now let's look at the comparative anatomy of the human genome and
its implications for evolution. As far as we know the genetic content ofthe X chromosome is identical in all mammals (20). Hemophilia is X-linked in man, dog and horse. Glucose-6-phosphate dehydrogenase is X-linked in many species, and so on. No exception is known to Ohno's Lawof the Evolutionary Conservatism of the X-chromosome. How about theautosomes? The banding pattern of the chromosomes in the higher apes,e.g. chimpanzee and orangutang, is strikingly similar to that of man (4,27). It therefore is not surprising that there is homology of synteny.(Synteny is a useful word introduced by James H. Renwick to mean "onthe same chromosome." Linkage means that they are close enough on
85
86 VICTOR A. MCKUSICK
the same chromosome to show lack of independent assortment. Twogenes can be syntenic and not be linked if they are sufficiently far aparton the same chromosome.) A good many genes that are syntenic in manare known to be syntenic also in the higher apes and to be situated onthe chromosome that is morphologically homologous.More surprising is the finding (Table 5) that there is considerable
homology of synteny in man's more remote relative, the mouse (17 ). Thisfact is being used as a clue to the chromosomal localization of genes inman. For example, in the mouse, albinism is linked to the beta hemoglobin
Human Chromosome
lp
1
3
4
6p
7
lop
10q
11
12p
15q
17q
19
21
TABLE VSyntenic Homology in Man and Mouse
Human Locus Mouse Locus
PGD PgdENO-1 Eno-1AK-2 Ak-2PGM-1 Pgm-2AMY-1 Amy-iAMY-2 Amy-2,fGAL-1 BgeACY-1 Acy-1PGM-2 Pgm-1PEP-S Pep-7ALB Alb-1HLA-A H-2DC4 SsHLA-B H-2KGLO-1 Glo-iGUS GusMDH-M Mor-1ASL AslPP PYPHK-1 Hk-1LIPA Lip-AGOT-S Got-1LDH-A Ldh-1Hb HbbTPI-1 Tpi-iGAPD GapdMPI Mpi-1PK-3 Pk-3GH dfTk-S Tk-1GALK GlkGPI Gpi-1PEP-D Pep-4SOD-1 Sod-lAVP AVP
Mouse Chromo-some
4
3
9
5
17
5
10
19
7
6
9
11
7
16
THE HUMAN GENOME
locus and one is thus justified in a strong suspicion that the albinismlocus in man may, like the beta hemoglobin locus, be situated on theshort arm of chromosome 11.Now for the functional anatomy of the human genome. It turns out
that the genes for the enzymes that catalyze successive steps in specificmetabolic pathways are not linked. It has been possible to look at this forfive different pathways (galactose metabolism, 3 enzymes; urea cycle, 3enzymes; tricarboxylic acid cycle, 5 enzymes; pentose phosphate pathway,3 enzymes; glycolysis, 9 enzymes), and it turns out (15, 16) that the genesfor the enzymes involved in each are widely scattered in the genome. Apossible exception is glycolysis where there is a suggestion of clusteringon the short arm of chromosome 12.Now for the developmental anatomy of the human genome. The
neatest example of apparent developmental significance of the arrange-ment of genes in a cluster is provided by the beta and alpha hemoglobingenes (Fig. 2). A sequential switching from epsilon to gamma to delta andbeta genes occurs from the 5' to the 3' end of this segment. The samething happens with the switch from zeta to alpha genes (which are closetogether on chromosome 16) during very early development.An exciting story is unfolding concerning the developmental anatomy
of the immunoglobin genes. Anatomic rearrangement apparently is im-portant during development and differentiation of the lymphocyte fordiversity of antibody production (26).Now for the applied anatomy of the human genome. Gene therapy
becomes ever a more realistic possibility, and knowledge of chromosomalanatomy will almost certainly be fundamental to that advance. Twoexamples of application of the anatomic information are already with us:in prenatal diagnosis and in premorbid diagnosis. This can be illustratedby two examples from the map of chromosome 6 (Fig. 4). The short armof chromosome 6 carries the major histocompatibility complex HLA withits four major components A, B, C, and D. One form of adrenal hyperpla-sia, 21-hydroxylase deficiency, is closely linked to the B locus (9, 12).Hemochromatosis is closely linked to the A locus (11). In a family inwhich 21-hydroxylase deficiency has occurred, the status of the unborninfant can be determined with high certainty from the HLA type of cellsobtained by amniocentesis, considered against the background of thefamily. Similarly, in a family in which hemochromatosis has been iden-tified, individuals homozygous for the gene, but as yet clinically unaf-fected, can be spotted by HLA type, and measures to limit iron load canbe taken in those persons before morbid change occurs in the liver,pancreas, or heart (5).The discussion to this point has concerned the nuclear genome, the sex
chromosomes and 22 autosomes. Man has a 25th chromosome, that of
87
VICTOR A. MCKUSICK
the mitochondrion. It is a single circular chromosome like that of bacteriaand determines certain enzymatic and probably structural elements ofthe mitochondrion (2). Mutation in mitochondrial genes may be the basisof some diseases (6); the only human mitochondrial mutation identifiedto date is one rendering cultured cells resistant to the effects of chlor-amphenicol (24). Mapping of man's 25th chromosome is also proceedingrapidly by the methods of molecular genetics.
Progress in the dissection of the human genome-or in the mapping ofthe human genome, depending on the metaphor you prefer-will, youcan be certain, be rapid in the last vintade of this century. Familymethods, somatic cell hybridization, and molecular genetics all have theircontributions to make, especially in combination. Family studies havehad a rebirth through the use of new polymorphic markers provided byrestriction enzyme analysis. Throughout the genome, both in coding andnoncoding parts, there are hereditary variations in what specific nucleo-tides are present, and as a result there are hereditary differences in theway the DNA is digested by restriction enzymes (10). Using such poly-morphic DNA markers in family studies (3) one can arrive at thechromosomal localization of conditions like achondroplasia, Marfan syn-drome, Huntington's chorea, myotonic dystrophy, hereditary polyposiscoli, and many others for which the basic biochemical defect is not knownand therefore no way to study the entity by somatic cell hybridization,for example.
Indeed, the technology is now available or within sight to determinethe full nucleotide sequence of the human genome within the near future,perhaps by the year 2000. Such a determination per se is unlikely to bea scientific priority. Even when the anatomy of the human genome isknown down to the last nucleotide, we will not know the function of allparts of that DNA, just as Sanger's classic description of the amino acidsequence of insulin did not provide any information on its function. Fullnucleotide sequencing of the human genome will be no mean task, asindicated by the fact that the diploid human cell has about six billionnucleotide pairs. One can generate numbers such as how long would ittake to sequence the genome if one nucleotide per second were identified.That number is more than 100 years for the haploid genome of a singleindividual. Collating the information on the human genome that I haveshown you here has been a cottage industry, or perhaps better, a stampcollecting operation. The library task of storing, collating and retrievingthe full nucleotide sequence of the human genome, including its varia-tions, is mind-boggling.This is a view of the human genome through the eyes of Mercator and
Vesalius, and yes, Mr. President, through the eyes of Captain Cook.
88
THE HUMAN GENOME 89
REFERENCES
1. Auerbach D, Bell GI, Rutter WJ, Shows TB: The insulin gene is located on chromosome11 in humans. Nature 286: 82, 1980
2. Beale G, Knowles J: Extranuclear Genetics. Baltimore. University Park Press, 19783. Botstein D, White RL, Skolnick M, Davis RW: Construction of a genetic linkage map
in man using restriction fragment length polymorphisms. Am J Hum Genet 32: 314-331, 1980
4. Dutrillaux B: Chromosomal evolution in primates: tentative phylogeny from Microcebusmurinus (Prosimian) to man. Hum Genet 48: 251-314, 1979
5. Edwards CQ, Cartwright GE, Skolnick MH, Amos DB: Homozygosity for hemochro-matosis: clinical manifestations. Ann Int Med 93: 519-525, 1980
6. Fine PEM: Mitochondrial inheritance and disease. Lancet II: 659-662, 19787. Francke U, Holmes LB, Atkins L, Riccardi VM: Aniridia-Wilms tumor association:
evidence for specific deletion of llpl3. Cytogenet Cell Genet 24: 185-192, 19798. Francke U, Oliver N: Quantitative analysis of high-resolution trypsin-giemsa bands onhuman prometaphase chromosomes. Hum Genet 45: 137-166, 1979
9. Glenthoj A, Nielsen MD, Starup J, Svejgaard A: HLA and congenital adrenal hyper-plasia due to 11-hydroxylase deficiency. Tissue Antigens 14: 181-182, 1979
10. Kan YW, Dozy AM: Polymorphism of DNA sequence adjacent to human beta-globinstructural gene: relationship to sickle mutation. Proc Nat Acad Sci 75: 5631-5635, 1978
11. Kravitz K, Skolnick M, Cannings C, Carmelli D, B Baty, Amos B, Johnson A, MendellN, Edwards C, Cartwright G: Genetic linkage between hereditary hemochromatosis andHLA. Am J Hum Genet 31: 601-619, 1979
12. Levine LS, Zachman M, New MI, Prader A, Pollack MS, O'Neill GJ, Yang SY, OberfieldSE, Dupont B: Genetic mapping of the 21-hydroxylase deficiency gene within the HLAlinkage group. New Eng J Med 299: 911-915, 1978
13. Lundin LG: Evolutionary conservation of large chromosomal segments reflected inmammalian gene maps. Clin Genet 16: 72-81, 1979
14. Maniatis T, Hardison RC, Lacy E, Lauer J, O'Connell C, Quon D, Sim GK, EfstradiatisA: The isolation of structural genes from libraries of eucaryotic DNA. Cell 15: 687-701,1978
15. McKusick VA: The anatomy of the human genome. Am J Med 69: 267-276, 198016. McKusick VA: The anatomy of the human genome. J Hered 71: 370-391, 198017. McKusick VA: Mendelian Inheritance in Man: Catalogs of Autosomal Dominant,
Autosomal Recessive and X-linked Phenotypes (ed 5). Baltimore, Johns Hopkins UnivPress, 1978
18. McKusick VA, Ruddle FH: The status of the gene map of the human chromosomes.Science 196: 390-405, 1977
19. Mumey N: Quartercentenary of the Publication of Scientific Anatomy (1543-1943).Denver, Range Press, 1944
20. Ohno S: Ancient linkage groups and frozen accidents. Nature 244: 259-262, 197321. Rowley JD: A new consistent chromosomal abnormality in chronic myelogenous
leukemia identified by quinacrine fluorescense and Giemsa staining. Nature 243: 290-293, 1973
22. Tooley RV: Some Portraits of Geographers and Other Persons Associated with Maps.London, The Map Collectors' Circle, 1975
23. Tooley RV: Title Pages From 16th to 19th Century. Map Collectors' Series, No. 107.London, Map Collectors' Circle, 1975, p 27
24. Wallace DC, Bunn CL, Eisenstadt JM: Cytoplasmic transfer of chloramphenicol resist-ance in human tissue culture cells. J Cell Biol 67: 174-188, 1975
90 VICTOR A. MCKUSICK
25. Wilson MG, Ebbin AJ, Towner JW, Spencer WH: Chromosomal abnormalities inpatients with retinoblastoma. Clin Genet 12: 1-8, 1977
26. Wilson R, Miller J, Storb U: Rearrangement of immunoglobulin genes. Biochemistry18: 5013-5021, 1979
27. Wisniewski LP, Witt ME, Ginsberg-Fellner F, Wilner J, Desnick RJ: Prader-Willisyndrome and a bisatellited derivative of chromosome 15. Clin Genet 18: 42-47, 1980
28. Yunis JJ, Sawyer JR, Dunham K: The striking resemblance of high-resolution G-banded chromosomes of man and chimpanzee. Science 208: 1145-1148, 1980
DISCUSSION
DR. ED WOOD (Philadelphia): Victor, we've been friends a long time so I hope you'llpermit me a slightly frivolous remark. I tried to do a rapid calculation and I think you'vemade another discovery: namely, at the rate of 25 mappings per year you may havediscovered a new meaning of tenure.
DR. BURROWS (Boston): You mentioned the prophylactic treatment of hemochromatosisin children, perhaps, of patients who can be identified as having the gene for hemochro-matosis. Do you have any idea about the time it might take for a clinical manifestation toappear-might it be twenty years? How do you think of this in terms of how soon or howlong in advance of the clinical manifestations treatment must be introduced?
DR. McKusIcK: I would refer you to the beautiful paper in the most recent Annals ofInternal Medicine, the senior author being Corwin Edwards who is one ofmy former houseofficers in Baltimore, now back in Salt Lake, working with Dr. Cartwright and others. Theythink the hemochromatosis gene is much more frequent than is generally realized. Ofcourse, women by virtue of a monthly bleed have a safety valve on iron loading so one halfof the people at risk are less likely to get into trouble. But they think in the first place it'srecessive. It's the homozygotes by and large who get into trouble. In the second place theyestimate that the frequency of the gene in Utah is about 6% and the frequency of thecarriers is 12 or 13%, and the frequency of homozygotes, that is the people getting intotrouble, is about 0.3 of 1%. They point out that in Brittany, which is the only other placewhere the frequency of the condition has been reliably identified, similar gene frequencieshave been found. Now, the main burden of the Annals paper is the description of theclinical and laboratory findings in the homozygotes which have been identified by thesegenetic methods. One can know that a given individual who's in a family that's beenascertained through an affected individual indeed is homozygous and thus they know thefull range of clinical severity and laboratory test abnormality, so you'll find your answerthere. There is plenty of time to practise preventive medicine in these individuals and tokeep them from getting iron overloading because the overloading is a process that builds upvery slowly over a period of years.