FEB 17 1964 SULDIARY OF ACTIVITIES AS A NATIONAL SCIENCE Fourn-troy SENIOR PCSSICCICRAL FELLCW Institute of Cenctics University of Pavia, Pavia, Italy Boward M. Cann, M.D. Fellowship Mo. 52066 In the period July 5, 1962 to July 5, 1963, I worked and studied at the testitute of Genetics, University of Pavis, italy, under the sponsorship of the Nationsi selence Foundation. I concentrated wy efforts, for the most part, in the statistical aspects of human genetics. Although I was not enrolled in any . forage) courses, I did attend English lengiage lecture courses given by N.T.J. ailoy (Stochas ti ie Processes) and M. Finura (lethensticel Population Genetics), w es well es numerous seminars. Uhile at ths Insticute of Cenetics, I worked on a number of projects which exe sumiczized Lelow: Vamophilia & fudy the mutations and cegregational basis of hemopailia A and B is belay studied. Spacial questionnaire forus designed to give pertinent genetical infsrestion about these 2 bleeding discases have been sent to nenzatologists in meny comtries, ond we have asked that they complete a fora on each of their patients with heme phiiia A oz B. Each form asks for the birth date of the patient, of his wother | ond father, and of his 4 grandparents; for the total nuwber of brothers and the uuber of brothers suffering from hemopliilia; for the presence or absence of tie carrier state in his sistera or materzal aunts 3 for the presence of hanophilia in maternal uncles or other relatives; for the age of onset of the menifest di- sease in the patient; and for the diagnosis (A or 2). | IE watation frequency increases with age, or if mutations are accumulated in ‘the gozete~producing organs with tine, on2 vould exeect an increased trans< wission of mutant-bearing genetes with increesing age of the parents. As the ege fi “NAY of parents at the tins of fertilisation increases, one would expect a rise in the frequency of carrier: of a uwutated gone (Lc. heterozygous at the partieul ar locus involwed) cmoug the offspriag. With rarpect to hemophilia A and B, which ara beth transmitted via the X chromosome, one might expect that ceses of mo fationsal origin would, on the averace, porcena at birth mothers of signtftecatly Sseater age than thoge cases which have arisen throuch segregation. The resson= tug is sinllar for materaal grandparonts in whom mutation is revedled by the baxth of an affected rrandson: these grandparents should possess mean ages, at birth of mothers cf patients, in excess of tzcee for maternal grandparents in wien the hemophilia ceus has segregated fron its normal allele. Further, if the sex-linked mutation has occurred in one of the maternal grandparents, on the everage, these relatives should be older at birth of the mother of the case being considered”. taan were the paternal grandparents at the birth of the patient's father, if mu- : tation accumulates with age. We are particularly interested in the contribution 3 of the maternal grandfather since the ucle eruetes are continually produced during the reproauctive years. In females, the gexetes are already produced, probebly by birta, end certainly by puberty. Thus, we ere looking for a maternal grendperental ege effect in cases of hemophilia from fomilies in which there exiate no history of the disease prior to tue generation of the patient, i.e. in those cases which conceivably could be of waternal grandpareatal mutetional origin. If our hypothesis is correct, we will see that, on the average, maternal grandparents were older than were paternal grandparents (used as controls) at the births of the patients' parents in these ecesea and slso older, on the average, then were paternal and maternal grandparents at births of parents of cases from families with a history of hemophilia prior to the patients" scenerations. . If mutation does accumilate with age of mothers, hemophiliecs from fazilies wita no other history of hemophilia (present or pest gencrations) will possess mothers whose average age et their birth is higher than that of mothers of patients with affected sibs or maternal relatives, other conditions being equal. oe To date, anne 220 forms have bean eouleted and returned: Eeratolegists in Ro. of Forms Hemophilia A Henophilia B Uaspecifiled Germany 64 23 , 2 NatherLlands 3 4 Sweden 16 2 Italy 12 i Arcentina SL 10 2 1 cacsn of a Siccasa which {8 not hemephilia hes else been received Eleven of these forms were rejectad because of information not coneistent with a dlaznosis of hemophilia A or B. Analyois of variance of mother's agza paxtitioned by presence or sheence of a faaily history of henophilia and by fauily (ucle sibship) size revealed no sige alficaat differance batween the averase ave of the mothers at birth of nonefamil Lal hemophiliacs aad that of the mothers et birth of pationts with a positive family history. Partitioning mother's age at birth of the patient eliminates cay bias feom the prepondsrance of sualler sire sitshiva in whale the acthers wlohe be exe pected to be younger than mothers of larver sibshizes. Even with the use of this partition there did not epsear to be any sigaifiicant difference aasng aces of mothers for the varicus olbslip sises encountered in this study. In general, wothers have bean youncer at birth of single non-feuilial cases than have been mothera at birth of patients whase sibs or maternal relatives are affected. A maternal craninsrental effect has not beon demonstrated. The following come parisons hava been mide: Fenilies with ao history of hemophilia in eaxlier generations (i.e. fanilies with noasibility that beuophilia fa caused from grancperental mitation) Age watoraal G.P, = Age paternal ¢.F. = X, Ags maternal G.M. - Age paternal G.H. = Xo “Age maternal Crandparants - Age paternal Grandparents = Ry ole In all threa covenxisoms, tha differences betvean the one of maternal ond paternal grandeparent{s) did mot sigaificently differ from zero, although for each comparison tha praternel grendparenti(s) was(were) older (on Che averaya). furthermore, each of the three age differences did not differ significantly feom 1.2 yeare, the meximun age difference voseible ecawuming that: —_ - pened nh — oe as Where X, = moan age of My + aX, fo maternal grandrerent in whoa mutation arises (or of cases M, ™ mutation rate at beginning - “ 9S * PRT of reproductive years with mutent gene). XK, sveraga age of control K, - %, will be mania when Hy 29 grendpercat. This value = 30 years (approximate) Thus: - 3 iecrease of mutent pro} -X = #62 2 aus XK, wf as 6 36/30 = 2.2 ak, RY portion of gemetes per year. | 62 w variance of gencration tine (or variance in age of grencparent at birth of mother affected. ‘This value = 36 years for English mothers (Penrose) it was also found that maternal grandfathers of cases from fanilieuw with no previous histery were slightly younger then maternal grandfathers from fantlies with a previous hensvalile history. Thus the behevier of the ages of grandparents or of mothers is uot what is exe scgtad with the hypothesio of mutation accumuletion. The expected effect may be + innonsteated with more cases. In thle type of study where the corzcct reporting of % fanily histery cad of birth dates is so crucial, one wonders what ths role of in- a couplets reporting pleys in the present cesusts. ~ oe these heuophiiia data are also being anciyned wita modern commuter techniques oi cegregation analysis, All families with ovo or more male sibs are being used for the anslysis. We are gtudying caly hemophilia A siace relatively few eases ei Lomephiiia B were reported. Corairy uesapadida A No. of Fanilies § & 2 Total Simplex Fanilies Multiplex Prailies vol r>y} Gesiiatay cud Phe Netherlands 29 #1 & Sweden 9 3 6 ztaly 3 1 Z Argentins 55 37 18 Total 96 62 34 Avcuug sepvegetion Erequenzy P o k (foc wedes) we heve estimated X (sporadic cave fvcquency in all affected cases} on the besie of 1) simgle selection (77, provenility of ascertalmieut, = 0) aud 2) truacete selection (7 = 1). Under cingle selection we estinate that X = .50 + .13 The parameters of P,77 end X have values which fit the cata for all countries reporting. Under truncate se- lsctioa, althoush x was estinated, the maziaun Likalikood ecore and varlence for p indicace an x" met cenpatible with the escumed valua of 0.5. Gur analyais is based oaly on the number of affected and unaffected sibs in eacn feally. The information about maternal uacles (affected and m Efectad) is mot cuaplete. it should ba noted that most of the forms from Germany do not con- tsan 4 question as to the muuber of affected male sibs; just the total musber of eile is Listed. We heve been assuning that the patient ie the only effected if chee ig co meation of other aficcted sibs. However, this may prove to be an un- \ wisvenlcd cesuaption upoa folleweup, which wist be cone. There appears to be a celicit of affected sibs in caces from Germany and Argentina, as indicated by LH) re wae hand teSulations in which the patient fe subtracted from the nuxhber of aja Effect of Selected Variebles on Secondary Sex Ratio Data from some 820,600 Italian birth certificates for Liveborn children and seme <0,0CO birth certificates for stillborn children for the year 1960 have been taculated and made available to us for analysis. ‘The data recorded for each birth contains the sex of the child, the age of the maternal grandfather at birth of the wITKEL, Che age of the mother at birth of the child, the order of birth represented by this birth, and the geographic region of birth. The object of the analysis was to look for an effect of maternal grandfather's age on sex Fatigein the population of births. If mutation aegumlates in the gamete pool with tine, and Lf ccometes ate actively produced curing the repreductive ases only in soles, sexelinked lethal | 5 recessive mutations should ggcur with increasing frequency as ee. Ancreases in the male, ‘Since a sexelinked lethal recessive mutation can only be expressed in the male grandchildren of the male in whom the mutation hes occurred, it is more Likely thet the mutation will be pacsed from older grandparents to mothers. The result of a sex-linked lethal recessive mutation in a population of births should be a deficit of males (or an increase in aborted male fetuses or stillborn male children). If mutation does accumlate with age, one would expect that the male-female ratio, or the proportion of males in a population of newborns, would tend to decrease as the ese of the maternal grandfather at birth of the mother increases. It is this effect which we cought in this study. We also investigated the effect of increasing mothers’ age (at birth of the child), the birth order, and tle geographic region of birth on the sex ratio of the population of 880,000 Italian liveborn children. Similar studies were carried out with the stillborn group. The sex ratio for all Italien livebirths in 1960 (M/F sex ratio) was 1.0553. For stillbirths the sex ratio was 1.2274, Whea the frequencies of male and female livebirths were distributed by the age of grendfathers at birth of the mothers, no heterogeneity of these data snong the —_" ages could be demonstrated by the chi square deteraination. Althougi no significant na aja sécveosion of these frequencies oa ages exicts, the regression coefficient ine cleated a peoltive trend for mules, i.e. a slight but nonsignificant increase of feequency of male nawborns with increasing age of maternal grandfatzcrs. No heterogeneity emoag etillbora male and femelle frequencies distributed smong creadfethers’ ages could be denonstrated end a recression coeffteteat wes found Whieh dndiented a ucgative (but aosalgnifieent)? trend of male stillbirths with fuerersing ara of grendfathers. To determine whether the lack of heterozenelty emong birth frequencies pare titioned by cex and distributed by ase of graadfathkers was duo to a peculiar cisteibution with respect to ome of the othss variables aveileble for analysis, a sisailer distrlsution (sex frequency by grandfathers’ aces) was made for eorh of mother at birth of the child, for exch order of birth (i to 8 of more) end for each gescrsphic region of birth. Seckl as beteregsnelty could be fouad for etiilborn or livebirth data distributions within each of the Susclasses Listed. Investigations of a possible effect of ose of mother at birth of tha child and of orcar of birth on tha sex ratio of Livsbora sad stitiborn parulatlons failed to demonstrate any ne taroganelty within distributions of sex frequencies for both variesles. Furthaor, ne heterogesetty was found smoaz the Livebirth sex ratio distributed by geographic vesion of birth. When the stillvirth population waa partitioned by esx and distributed by geo- grepnic region, however, a highly sigulfieant hateregenaity was found xsoug the cox ratios. Furthetuore it wes found that tha manle stillbirth frequencies shoved a positive end slenificant vegression on geesrachle region in a roushly north to outh azis. When Sardecua and Sicilia were omitted from the eciculation, the ree gvossion became mora sisuificant ead more positive. The values sivea to the indee poncont variable, the regions, ware far fron rrotice, being successive intesers frea i through li for the eleven regional clessifications, progressing fzom north to couta, used by the Italian Istituto Centrale Di Statistica. To improve the pre- eisdcn of the representation of Che independent variable, the acillbirkh rate per ty ae (LOTS tetal bioths, computed frou the data for each cecdom, was used. fhe stillbirch faz ues found to ronuchly fueveece fron northern Chrouch southern Itelien restona. Consntation of weishted end umeighted ragressioms ravenled in each instante 2 sige nitienstly positive regression of freavency of male stillbirths on incressinag te- gicusi atillbirth rate. Socloecononie conditions decline, ia general, the further seul. one pregeesoes in Itely, aad this decline could explain the iuerease Im still- - : “4 Darth Tate. Uovever, it is ciffieult to imelicate « cetertorating sociocconenic euviromywat iu altering tle gtillbirth sex ratlo beyond suck vague statements as male Setucca, tera and mar term, seem more susceptible to an unfavorable envirormarut. than co their fennle countergper ts. Agmutination of iru Rec Plead Cells by Mouse Toomitinesa ae wt a Us have found ayglutLasting astivity fer Inwun ved bleed ceils in @ nusber of waoase isocatisera. If thls cetivity is antibody, the devalopment end enhancencnt anacica sy clivmaln or with trypsinised erythyosytes suggests that it fs sine diss to duesiuplate eatibody. Since all erythrocytes so far tested react with positive iscongisera, it appears that the agglutinatisa is modiated by aatizen otucr than A, > 3, O, tae uzual Rh comgonuents, M or NH, © possibly aa unimows erythrocyte antigen. We hope ¢3 sexeca red bleed cells from usny donors in an atteupt te identify the esntigen saould this activity be en imme response te mause tissue antigunse, the inrmuno« gen is protebly an entisenuis conmples and is certatuly not Limited to H - 2 eutigen. Genetic disebailarity ef the tiesue donor and the eulmal being imumiced for autizverun prodvection seams te be lavertent for strong nuuan hemagglutinating activ fey this relutionship cas be invasticated further by testing antisera of various conorehost 4.2 Rm CEU ANLELOUS » Low exclutiastion ticera in posled, noraal mouse serum mazes it conceivable thet ee s activity i3 not aa bors me response, belas exhaused cola¢ identally with or indivectly -j- ty isoimunization. Testing of individual ssxples of normal serum from animals of & veelety of strains way contribute to further characterization. We misht ecnsider i* tid i + wacling of mice may i:xmmice them to human aatizens, but the high titers of sgglutinating activity im pesttive sera suggest an iccumization process of greater magaituds then skin to skin contect. Absorption of the agrlutiucting ability of toonntisera by Ivenen exytoresytes is not direst proof of antibody but is so.zwhat reaseuring., By altering various fac- tera (2.9. erythrocyte entigenic make up, numher of cells used, number of 2bserptions) in future absorptions, 4t may be neselble to cenoastrate more meaningful differential absorptions. Finakly, 1t will be iuportsat to determine 1f various mouse ontigens ean zbsorb the kuman hencggluttasting activity. Eéfact of Month of Birth on Selected Variable for Conserinta Recent stucics have indicated the pesaibilicy of a relationship betwean the aonth of birth and the developaant of mintel retardation. Fnobloch and Pasaccuick analyzed birth dates of indiyidusls admitted to a state school for meatelly retarded chiléxen in the period 1912-1248. These authers found that significrntly more had been bora in the months Jenuacy, February ord Merch. duding ond explove related pheaonena, we have he z 3 ety In an atteape to elucicnte th g undertaken a survey in which the effeet of the month of birth on a number of variables is being determined. The study so fer hes casit. with varissles drown from the records of a sroup of conscripts fron the rural ervecs in the Province of Purse. These records give information pertrining to the medical history, physicel cxcaination, socLoeconomic status, education and vital statistics, including birta date, of ecsh consexipt for the period 1500-1910. The vccords have been ebstracted, cole’ end punched onto I.B.M. a cards. The plan of the etudy was otraisutforvard. A number of variables available on punched cards were selected and each variable was distributed by the months of birth of the conscripts. This task was perforsed using I.3.M. sorting and tabulating machin- exy. A relationship between each variable ead the moath of birth was sought by estesuining 1£ heterogeneity of frequencies anong the birth months existed. Chi cyuare was used to test for heterogeneity and was determined by the electronic cone puter for each verlabie. Finally using only those varfiebles which showed hetcro- geneity enong birth months, a sine curve (serlodic regression) regression wes calcu- lated for cach variable subclecetfication uyon month of birth and the sionificeace of txe Geviatioan of the regression curve from the mean determined by the analysie of vorisres. The electronic cowuputer was ecpleyed for these calculations. whe variables selected from the conscripts" records were: Varieble Subclassification ; WKN Table Milltary fitness. Accepted, ‘Parital’ Rejection 3X12 Rejection (oa wedical basis) Reasons for Rejection Possible Mental Deficilency*, Others 2X12 = (*cerebropatnaies, epilepsy, meatal Giseases, deaf-iute) “Hair Color - Blond, Bed, Brown, Biack | &K12 Generel Pigmentatioa Light Wealthy, Pale, Dark Healthy : 3X12 General Picmantation Light Healthy and Pale, Dark Healthy 2X12 Genersl Pigmentation Light Healtzy, Dark Ecalthy 2X12 Occupation . | Ferner, Non-Farmer 2X12 Literacy Literate, Partially Literate end 2x12 Illiterate Literacy Literate end Partislly Literate, 242 Tiliterate Georraphic Zone of Each of the 1i zones of residence 11 Xk 12 at the: of conscription Heterogeneity of frequencies among birth months was found for the following varisbles: general pigmentation, literacy end seographic zone of residence. Ail other vatiebles were distribédted homosencously c~ong the 12 months. ‘ts pertodic regreasioa sine curves cciculated from the data for general pignen- tation for recruits classified as Light Hesithy and as Dark Healthy daviated signifi- ? wise a Baw eg: a. am . 7 ad a at COAL EN =, - ‘ a 4 a onchy fvaa tha ganetal moan (p< 0.05). the sreatsct Leaquency of birth of “igh ft sytem Oo eB eb gat ~ . er ce aqesen ey 5? a an aa rags 5 . . Vaclohg" voarudts ceourved tn Septewler, The frequecey then coclined to the lcvest oi fe pak ft “ a ats wag ; a t. 4 rime which oceur fa March aud then began to admcrenre, A raeiprocal behavior GE the eine cutyo wea Zound for more haavily pipecated ceeruite, the highest fvequency of births of thease recruits cceurring in March and April and the lowest frequency in iu the ease of Literccy, Literate ricralte were bern wlth the highest frequency n thas month of Novarcer, the moaths of lovest fgequemey being April and Ney. The pariedic rerression curwe for Literate veacvuits mad for pooled partially Litexate and Literate recruits doviated significantly from the prand mean. The eine curves commuted from freauencies of month of birth of recruits from the 4 ceogvenhie zomes censisting of comemea Colorno, Mezzani, Sorbolo, Torxile, Fornoro, Garo, Pellecrine, Varano, Maleseari, Covaighic, Monchio delle Cor, Pelencano, Tissano, Folino and Sala Pajense deviated significantly from © the means. Tha hichest birth free Fed queactes for recruite from Colorno, Megzani, Sorbele, Torrile, Felino ond Sails Bagenza occurred i2 Cetobser while the Lowant occurred in April. For racruits from Cozniglio, Mouchio delle Cor, Polansevo, Tizsano, Fotaevs Tera, Pellegeina and Varcno Nelegari, | the hishkest birth framieacies occurred in the spring monthe wate the Loz: aot occurred im Auten and early wint All sine curve computations wore made with relative fvequencies transformed to radians (evesin Vp) so that the vasression values aca not biased by the monthly birth frcquancies for ali the vecrulta. The pexvlodic affeet of month of birth on Literacy and secgr epaie cone of residence io probebly related to seaisacononie factors, while tha af slouantation may Ladicate the iupest ef physical or biological eavirommcut on the developing embryo end fetus. | | Further etudiea of the affect exavted ty the month of birth axe indicated. It is. tovsrn thet isoantibedy titers vary poriodiczily ia cattle and man. An fuvestigetion 4, of the periodicity of ischeusgglutinin titers in asa with special eaghasis on phenomena Phe ty ol poucckhly welated to dnmcousatibility (sush variclias sa miscarriases, desmtitedy Leos GL fava, Gceurrenca of Ricelyete Clecase of tha noviern being studied) is pertisularly ame * wpe pects o® o. aye tan a! a . oo fe ; eaedly erhilewed., Phere ere sow iudiesctons that the sex retio im usa wie could well Le the result of cocdioeconente bias intwoeciead by thea aethed of Gata collection, Tt will be worthwhile to expler 7 * q $0 Vey + . rates a Sa wo ~ ~~ ees ag yea Cetes2 the valatlonship between sen ratio sud month of birth. Secrocation Anelyeis of Distetes Mellitus, Juvenile Type % Tsang Gates collected fer a genetic stity of juvenile diabetes mellitus (Sicpsen, WR. Annals of Mimaa Genatles (1962) 26, 1} - otity of the genetic narc:ctcuvs Lavolved y & ? was carried out. Tha seprezetion analysis techniques of Morton were employed with the electronic comsutar, The hypothesis of sx, outesonunl xeecesive single locus inkeriten ie wes teeted, The data contaiued 2 grovp of mabings which were possibly intarerosses Cua affected paventa, one or wore affected offspring) anc genother group of satiags which were poceibhle brackerossee (one affected parent, one of wore aifag te G offaprin;3. ate ana year which I spent et the Institute of Canctics was an enrichins experience end has ceatributed to cy carcer ia academic Pediatrics and research and study in heritsble disezse.