Allelic association discriminates draft orders

A year ago there was hope that a finished sequence of the human genome would soon be publicly available and would give a more reliable locus order than an unconstrained radiation hybrid or genetic map. Alas, there are now different draft orders for each region, none of which may be correct because of gaps, uncertain polarity of contigs, and errors in assembly. Shortly before these drafts became available, we analysed allelic association (also called linkage disequilibrium, LD) in the FRAX region in a large sample of haplotypes (Ennis et al . 2000). We demonstrate here that this material discriminates among alternative draft orders. To express support for discrimination between two values of χ²=−2 ln L we use the Akaike criterion AIC = df[χ²/min χ²−1]. Excluding premutations and full mutations at FMR1, all maps have 715 degrees of freedom (df) among 717 pairs of alleles after accepting L = 0 and estimating M, ∈ in the Malecot equation E(ρ) = Me ^−∈d, where ρ is the association between a pair of alleles at distance d. An AIC in excess of 2 provides evidence against a map with the larger χ².     

Increased glomerular angiotensin II binding in rats exposed to a maternal low protein diet in utero

In the rat, protein restriction during pregnancy increases offspring blood pressure by 20–30 mmHg. We have shown in an earlier study that this is associated with a reduction in nephron number and increased glomerular sensitivity to angiotensin II (Ang II) in vivo . Hence, we hypothesized that exposure to a maternal low-protein diet increases glomerular Ang II AT₁ receptor expression and decreases AT₂ receptor expression. To test this hypothesis, pregnant Wistar rats were fed isocalorific diets containing either 18% (control) or 9% (LP) protein from conception until birth. At 4 weeks of age, the kidneys of male offspring were harvested to measure cortical AT₁ and AT₂ receptor expression, ¹²⁵I-Ang II glomerular binding, tissue renin activity, tissue Ang II and plasma aldosterone concentrations. AT₁ receptor expression was increased (62%) and AT₂ expression was decreased (35%) in LP rats. Maximum ¹²⁵I-Ang II (¹²⁵I-Ang II) binding ( B _max) was increased in LP rats (control n = 9, 291.6 ± 27.4 versus LP n = 7, 445.7 ± 27.4 fmol (mg glomerular protein)⁻¹, P < 0.01), but affinity ( K _D) was not statistically different from controls (control 2.87 ± 0.85 versus LP 0.84 ± 0.20 pmol ¹²⁵I-Ang II, P = 0.059). Renal renin activity, tissue Ang II and plasma aldosterone concentrations did not differ between control and LP rats. Increased AT₁ receptor expression in LP rat kidneys is consistent with greater haemodynamic sensitivity to Ang II in vivo . This may result in an inappropriate reduction in glomerular filtration rate, salt and water retention, and an increase in blood pressure.     

Association of HincII RFLP of low density lipoprotein receptor gene with obesity in essential hypertensives

Obese (BMI 26 kg/m²; n=51) and lean (BMI <26 kg/m²; n=61) Caucasian patients with severe, familial essential hypertension, were compared with respect to genotype and allele frequencies of a Hin cII RFLP of the low density lipoprotein receptor gene ( LDLR ). A similar analysis was performed in obese (n=28) and lean (n=68) nonmotensives. A significant association of the C allele of the T → C variant responsible for this RFLP was seen with obesity ( x ²=4.6, P =0.029) in the hypertensive, but not in the normotensive, group (odds ratio=3.0 for the CC genotype and 2.7 for CT ). Furthermore, BMI tracked with genotypes of this allele in the hypertensives ( P =0.046). No significant genotypic relationship was apparent for plasma lipids. Significant linkage disequilibrium was, moreover, noted between the Hin cII RFLP and an Apa LI RFLP ( x ²=33, P <0.0005) that has previously shown even stronger association with obesity (odds ratio 19.6 for cases homozygous for the susceptibility allele and 15.2 for heterozygotes). The present study therefore adds to our previous evidence implicating LDLR as a locus for obesity in patients with essential hypertension.     

Angiotensin II-induced modulation of endothelium-dependent relaxation in rabbit mesenteric resistance arteries

The role of local endogenous angiotensin II (Ang II) in endothelial function in resistance arteries was investigated using rabbit mesenteric resistance arteries. First, the presence of immunoreactive Ang II together with Ang II type-1 receptor (AT₁R) and angiotensin converting enzyme (ACE) was confirmed in these arteries. In endothelium-intact strips, the AT₁R-blocker olmesartan (1 μ m ) and the ACE-inhibitor temocaprilat (1 μ m ) each enhanced the ACh (0.03 μ m )-induced relaxation during the contraction induced by noradrenaline (NA, 10 μ m ). Similar effects were obtained using CV-11974 (another AT₁R blocker) and enalaprilat (another ACE inhibitor). The nitric-oxide-synthase inhibitor N ^G-nitro- l -arginine ( l -NNA) abolished the above effect of olmesartan. In endothelium-denuded strips, olmesartan enhanced the relaxation induced by the NO donor NOC-7 (10 n m ). Olmesartan had no effect on cGMP production (1) in endothelium-intact strips (in the absence or presence of ACh) or (2) in endothelium-denuded strips (in the absence or presence of NOC-7). In β-escin-skinned strips, 8-bromoguanosine 3',5' cyclic monophosphate (8-Br-cGMP, 0.01–1 μ m ) concentration dependently inhibited the contractions induced (a) by 0.3 μ m Ca²⁺ in the presence of NA+GTP and (b) by 0.2 μ m Ca²⁺+GTPγS. Olmesartan significantly enhanced, while Ang II (0.1 n m ) significantly inhibited, the 8-Br-cGMP-induced relaxation. We propose the novel hypothesis that in these arteries, Ang II localized within smooth muscle cells activates AT₁Rs and inhibits ACh-induced, endothelium-dependent relaxation at least partly by inhibiting the action of cGMP on these cells.     

Donor-recipient sharing of HLA class II alleles predicts earlier recurrence and accelerated progression of hepatitis C following liver transplantation

Abstract: Both direct viral cytopathic effects and host immune responses appear to be important in the pathogenesis of hepatitis C virus (HCV) infection. Liver transplantation provides a means to explore the role of the immune system in the development of HCV-related liver damage through comparing the natural history of HCV in patients with different degrees of donor-recipient human leukocyte antigen (HLA) matching. We evaluated 36 patients with recurrent hepatitis C viremia following liver transplantation to determine whether hepatocellular injury or progression to bridging fibrosis occur more rapidly when donor and recipients share HLA alleles. HLA typing for the HLA-A and HLA-B loci was performed by serological techniques and PCR-based oligotyping was used to type alleles of the DRB1, DRB3, DQA1, and DQB1 loci. A median of eight liver biopsies, obtained during a median follow-up of 36 months, were reviewed per patient. Donor-recipient sharing of alleles of HLA-DQB1 or DRB1 was associated with more rapid development of recurrent hepatitis by univariate analysis (χ²=5.7, P =0.02 and χ²=5.54, P =0.02 respectively). However, only sharing of HLA-DRB1 alleles was identified as an independent predictor of reduced time to recurrent histologic injury by multivariate analysis (χ² =5.74, P = 0.02). Furthermore, sharing of HLA-DRB3 and histologic evidence of rejection were associated with more rapid progression to bridging fibrosis both by univariate methods (χ²=4.12, P =0.04 and χ²=4.66, P =0.03 respectively), and by multivariate analysis (χ²= 13.01, P =0.001). These findings suggest that HLA class II-restricted immune responses may contribute to the pathogenesis of HCV-related liver injury in liver transplant recipients.     

Hidden linkage: a comparison of the affected sib pair (ASP) test and transmission/disequilibrium test (TDT)

I compare the transmission/disequilibrium test (TDT) and affected sib pair (ASP) test under a general algebraic model describing a bi-allelic disease locus. Assuming linkage to a bi-allelic marker, I derive two binomial probabilities, one for parental allele 'transmission' (Pt ) which determines the magnitude of the TDT χ² statistic (χ²_tdt), and a second for identity-by-descent (ibd) marker allele 'sharing' ( P _s) which determines the magnitude of the ASP test statistic (χ²_asp). I also consider the ASP test applied to a completely polymorphic marker and demonstrate that the probability of ASP marker allele sharing ( P _s) is identical to P _s observed for a bi-allelic marker in equilibrium with the disease locus. I present a general framework for determining the power of the TDT and ASP test based on expressions for P _t, P _s and the proportion ( H / F ) of ascertained parents who are informative at the marker. Two previous analytic investigations of TDT power based on the work of Ott (1989), and Risch & Merikangas (1996) are shown to be special cases of this general framework. In addition, I show the relationship between the framework I present and a third analytic investigation of TDT power for multi-allelic markers based on the work of Sham & Curtis (1995).     

Angiotensin II: a major regulator of subcutaneous adipose tissue blood flow in humans

We investigated the functional roles of circulating and locally produced angiotensin II (Ang II) in fasting and postprandial adipose tissue blood flow (ATBF) regulation and examined the interaction between Ang II and nitric oxide (NO) in ATBF regulation. Local effects of the pharmacological agents (or contralateral saline) on ATBF, measured with ¹³³Xe wash-out, were assessed using the recently developed microinfusion technique. Fasting and postprandial (75 g glucose challenge) ATBF regulation was investigated in nine lean healthy subjects (age, 29 ± 3 years; BMI, 23.4 ± 0.7 kg m⁻²) using local Ang II stimulation, Ang II type 1 (AT₁) receptor blockade, and angiotensin-converting enzyme (ACE) inhibition. Furthermore, NO synthase (NOS) blockade alone and in combination with AT₁ receptor blockade was used to examine the interaction between Ang II and NO. Ang II induced a dose-dependent decrease in ATBF (10⁻⁹ m : −16%, P = 0.04; 10⁻⁷ m : −33%, P < 0.01; 10⁻⁵ m : −53% P < 0.01). Fasting ATBF was not affected by ACE inhibition, but was increased by ∼55% ( P < 0.01) by AT₁ receptor blockade. NOS blockade induced a ∼30% ( P = 0.001) decrease in fasting ATBF. Combined AT₁ receptor and NOS blockade increased ATBF by ∼40% ( P = 0.003). ACE inhibition and AT₁ receptor blockade did not affect the postprandial increase in ATBF. We therefore conclude that circulating Ang II is a major regulator of fasting ATBF, and a major proportion of the Ang II-induced decrease in ATBF is NO independent. Locally produced Ang II does not appear to regulate ATBF. Ang II appears to have no major effect on the postprandial enhancement of ATBF.     

Prenatal corticosterone exposure results in altered AT1/AT2, nephron deficit and hypertension in the rat offspring

Maternal treatment with the synthetic glucocorticoid, dexamethasone has been reported to result in a nephron deficit and development of hypertension in the offspring of rats. However, it is not known whether elevated maternal corticosterone (CORT), the natural glucocorticoid, has similar effects on blood pressure and nephron endowment. The present study investigated the effects of CORT (0.8 mg kg⁻¹ day⁻¹) administration on embryonic day 14 (E14) and E15 of pregnancy on: (1) nephron number at postnatal day 30 (PN30); (2) blood pressure at PN120; and (3) receptors of the renal renin–angiotensin system (RRAS) (AT₁Ra, AT₁Rb and AT₂Ra) during both embryonic (E16, E20) and adolescent (PN30) life. Plasma CORT concentrations were approximately doubled 30 min after injection. Unbiased stereological analysis revealed that maternal CORT treatment resulted in a nephron deficit of 21 and 19% in male and female offspring, respectively. Mean arterial pressures were significantly elevated in offspring of both sexes from the CORT group. Real-time PCR revealed that CORT treatment increased expression of AT₁Ra and AT₂R at E16, and at PN30. Expression of AT₁Rb was downregulated in embryonic life but upregulated at PN30. We believe that these results are the first to demonstrate that maternal CORT treatment results in a nephron deficit and development of hypertension in the rat offspring. Changes in the RRAS may be contributing to these phenotypes. Critically, this study suggests that increased but physiological levels of the natural glucocorticoid can programme similar changes to those seen with pharmacological doses of the synthetic glucocorticoid. This may have important implications for women experiencing significant stress during pregnancy.     

Sequence variants in the 5' flanking region of the leptin gene are associated with obesity in women

Few mutations have been found in the human leptin gene and the relationship between leptin gene sequence variation and human overweight is uncertain. To determine whether sequence variation within the leptin gene and its regulatory elements contribute to extreme obesity, we screened ∼3 kb of the 5' flanking region and the three exons in 125 unrelated extremely obese (BMI ≥ 40 kg/m²) and 86 average weight women (BMI < 27 kg/m²). Within the protein coding regions only one heterozygous silent mutation was found (codon 102; AAC/AAT). Within the 5' flanking region, six frequent sequence variants were detected ( q > 0.10), and the allele frequencies of three of these variants differed between obese and average weight Caucasian women (+19, χ²= 4.46, p = 0.035; −1823, χ²= 4.36, p = 0.037; −2548, χ²= 5.73, p = 0.017). Nine infrequent sequence variants were detected ( q < 0.05) but they did not occur more often among obese women compared with those of average-weight. For extremely obese women, three polymorphisms (+19, −188, and −633) predicted the degree of obesity. Allelic variants may influence the regulation of the leptin gene and thereby influence body weight, particularly among extremely obese women. However, given the low variability in coding regions and the high variability in the 5' flanking region, discerning the functional significance of each variant is likely to be difficult.     

The angiotensin receptor type 1-Gq protein-phosphatidyl inositol phospholipase Cbeta-protein kinase C pathway is involved in activation of proximal tubule Na+-ATPase activity by angiotensin(1-7) in pig kidneys

In a previous study, we observed that angiotensin(1–7) (Ang(1–7)) stimulates proximal tubule Na⁺-ATPase activity through the angiotensin receptor type 1 (AT₁R). Here we aimed to study the signalling pathways involved. Our results show that the stimulatory effect of Ang(1–7) on Na⁺-ATPase activity through AT₁R involves a G_q protein–phosphatidyl inositol-phospholipase Cβ(PI-PLCβ) pathway because: (1) the effect was reversed by GDPβS, a non-hydrolysable GDP analogue, and by a monoclonal G_q protein antibody; (2) the effect was similar and not additive to that of GTPγS, a non-hydrolysable GTP analogue; (3) Ang(1–7) induced a rapid decrease (30 s) in phosphatidylinositol 4,5-bisphosphate levels, a PI-PLCβ substrate; and (4) U73122, a specific inhibitor of PI-PLCβ, abolished Ang(1–7)-induced stimulation of Na⁺-ATPase activity. Angiotensin(1–7) increased the protein kinase C (PKC) activity similarly to phorbol-12-myristate-13-acetate (PMA), an activator of PKC. This effect was reversed by losartan, a specific antagonist of AT₁R. The stimulatory effects of Ang(1–7) and PMA on Na⁺-ATPase activity are similar, non-additive and reversed by calphostin C, a specific inhibitor of PKC. A catalytic subunit of PKC (PKC-M) increased the Na⁺-ATPase activity. These data show that Ang(1–7) stimulates Na⁺-ATPase activity through the AT₁R–G_q protein–PI-PLCβ–PKC pathway. This effect is similar to that described for angiotensin II, showing for the first time that these compounds could have similar effects in the renal system.     

Serotonergic 5-HT1a Receptors Regulate a Cell Contact-Mediated Interaction between Natural Killer Cells and Monocytes

Autologous monocytes irreversibly suppressed functions of human natural killer (NK) cells including baseline and lymphokine-induced cytotoxicity, antibody-dependent cellular cytotoxicity (ADCC). and interleukin-2 (IL-2)-induced proliferation. The suppression of these NK-cell functions was cell contact-dependent and could be evoked only by purified monocytes, recovered directly from peripheral blood by countercurrent centrifugal elutriation (CCE). The presence of monocytes also induced the disappearance of CD16 and CD56 antigen on CD3^- NK cells (CD3^-/16 ⁺/56⁺ CD3^-/16^-/56^-). By contrast, T-cell proliferation and the expression of CD3 on CD56^- T cells were not susceptible to cell contact-mediated suppression by monocytes. The biogenic amine serotonin abrogated monocyte-induced suppression of NK-cell functions as well as down-modulation of CD16/56 NK-cell antigen. Serotonin thus markedly augmented baseline and lymphokine-induced NK-cell cytotoxicity. ADCC, and NK-cell proliferation, and maintained the expression of NK-cell surface antigens in the presence of elutriated monocytes. The effect of serotonin was mediated by 5-HT_1A-type serotonin receptors (5-HT₁aR) as indicated by mimicry exerted by 5-HT_1AR ugonists such as 8-OH-DPAT and (+)-ALK, partial antagonism by the 5-HT_1AR antagonists pindolol and cyproheptadine. and lack of antagonism by the 5-HT₂R antagonist ketanserin or the 5-HT₃R antagonist ondansetron. Our data are suggestive of a cell-lo-cell-mediated mechanism by which monocytes down-modulate NK-cell function and phenotype and its serotonergic regulation.     

Chi-squared tests with small numbers

The usual advice given in standard texts is to combine classes when expected numbers are too small. It is suggested that an alternative would be to use the exact values of teh mean and variance of χ², for which algebraic expressions are provided. The reliability of the test can be improved by using √χ² instead of χ² as the test criterion.     

Histaminergic Regulation of NK-Cells: Protection Against Monocyte-Induced Apoptosis

Human natural killer (NK) cells (with CD3⁻/56⁺ phenotype) acquired features characteristic of apoptosis after incubation with autologous monocytes, as revealed by apoptotic nuclear morphology and degradation of DNA into oligonucleosomal fragments. The monocyte-induced apoptosis in NK-cells was prevented by the biogenic amine histamine at concentrations exceeding 0.1 μ M . The protective effect of histamine was blocked by the H₂-receptor (H₂R) antagonist ranitidine but not by AH202399 A, a chemical control to ranitidine devoid of H₂R affinity. It is concluded that histaminergic mechanisms may serve to protect NK cells from damage inflicted by products of the oxidative metabolism of monocytes.     

Family and population studies of SAHH and ADA polymorphisms. A possible pitfall in the ascertainment of SAHH electrophoretic phenotypes

Typing of both SAHH and ADA red cell electrophoretic patterns was carried out among the members of about 80 families from Latium (Central Italy) and in a random sample of about 350 individuals from two Italian regions, Latium and Sardinia.
1. The SAHH ¹ enzyme product provided another interesting example of a change in the electrophoretic pattern brought about by the haemolysate ageing. In vitro storage of SAHH 1 red cell lysates leads to the production of a pattern similar to that expected from a heterozygote SAHH 2–1. This change has been shown to be abolished by pretreating the sample with mercaptoethanol.
The results indicate that the systematic use of sulphydril reducing agents can provide a more reliable means of analysing the SAHH polymorphism if differently stored samples are to be compared by starch gel electrophoresis.
2. Evidence against complete linkage of the SAHH and ADA loci has been obtained from two informative SAHH/ADA matings encountered in this study.
3. The SAHH allele frequencies observed in the two samples analysed were: SAHH ¹= 0.969, SAHH ²= 0.024, SAHH ³= 0.007 (Latium) and SAHH ¹= 0.973, SAHH ²= 0.011, SAHH ³= 0.016 (Sardinia).
4. The ADA ² allele frequency estimates were: 0.083 (Latium) and 0.059 (Sardinia). These figures are almost identical to those already reported for the same two regions.     

Renal (pro)renin receptor upregulation in diabetic rats through enhanced angiotensin AT1 receptor and NADPH oxidase activity

Recent studies have demonstrated the presence of the (pro)renin receptor (PRR) in the glomerular mesangium and the subendothelial layer of the renal arteries. We hypothesized that diabetes upregulates PRR expression through enhanced angiotensin subtype 1 (AT₁) receptor–NADPH oxidase cascade activity. Using real-time polymerase chain reaction, Western blot analysis and immunostaining, we studied renal localization of the PRR in the streptozotocin-induced diabetic rat model and in response to 1 week of treatment with the AT₁ receptor blocker valsartan (10 mg kg⁻¹ day⁻¹), the angiotensin AT₂ receptor blocker PD123319 (0.5 mg kg⁻¹ day⁻¹) or the NADPH oxidase inhibitor diphenylene iodonium (DPI; 0.5 mg kg⁻¹ day⁻¹) 6 weeks post-induction of diabetes. Both PRR mRNA and protein were expressed constitutively in the kidneys of normal rat renal cortex and medulla, mainly in glomerular mesangium, proximal, distal and collecting tubules. Compared with normal rats (100%), diabetic rats demonstrated an increase in renal PRR mRNA (184%), protein (228%) and immunostaining. Valsartan and DPI prevented the increase in the PRR mRNA (106 and 126%, respectively), protein (97 and 140%, respectively) and immunostaining that was seen in the kidneys of diabetic rats. The AT₂ blocker PD123319 did not have significant effects on PRR mRNA (157%) or protein expression (200%) in the kidneys of diabetic rats. These results demonstrate that the PRR is constitutively expressed in renal glomeruli and tubules. Expression of the PRR is upregulated in diabetes via enhancement of AT₁ receptor–NADPH oxidase activity.     

Selfish prion of Rnq1 mutant in yeast

[ PIN ⁺] is a prion form of Rnq1 in Saccharomyces cerevisiae and is necessary for the de novo induction of a second prion, [ PSI ⁺]. We previously isolated a truncated form of Rnq1, named Rnq1Δ100, as a [ PSI ⁺]-eliminating factor in S. cerevisiae . Rnq1Δ100 deletes the N-terminal non-prion domain of Rnq1, and eliminates [ PSI ⁺] in [ PIN ⁺] yeast. Here we found that [ PIN ⁺] is transmissible to Rnq1Δ100 in the absence of full-length Rnq1, forming a novel prion variant [ RNQ1Δ100 ⁺]. [ RNQ1Δ100 ⁺] has similar [ PIN ⁺] properties as it stimulates the de novo induction of [ PSI ⁺] and is eliminated by the null hsp104Δ mutation, but not by Hsp104 overproduction. In contrast, [ RNQ1Δ100 ⁺] inherits the inhibitory activity and hampers the maintenance of [ PSI ⁺] though less efficiently than [ PIN ⁺] made of Rnq1–Rnq1Δ100 co-aggregates. Interestingly, [ RNQ1Δ100 ⁺] prion was eliminated by de novo [ PSI ⁺] induction. Thus, the [ RNQ1Δ100 ⁺] prion demonstrates selfish activity to eliminate a heterologous prion in S. cerevisiae , showing the first instance of a selfish prion variant in living organisms.     

Different break-points in Philadelphia chromosome variant translocation and in constitutional and sporadic translocations

Three different samples of translocations were considered in an attempt to identify those chromosomal bands preferentially involved in variant Philadelphia chromosome (PH¹) translocations, and to compare them with bands preferentially broken in constitutional and sporadic translocations. The first sample included 204 cases of variant Ph¹ translocations with 221 identified break-points (bp), the second consisted of 106 cases of non-Robertsonian constitutional translocations with 213 bp identified, and the third one of 185 bp identified in sporadic translocations found occasionally in single cells of subjects with normal karyotypes and without haematological disorders.
A statistical analysis demonstrated that there are some bands preferentially broken in each of the samples, and this with a high level of significance ( p < 0.001). The analysis of the distributions of the χ² components permitted us to identify the 12 bands preferentially involved in variant Ph¹ translocations and the 13 and 9 bands preferentially involved in constitutional and sporadic translocations, respectively. The comparison among the groups of preferential bp showed that the bands most involved in the three samples are different.
Some theoretical problems related to the origin of the Ph¹ chromosome are discussed.     

CTLA-4 +49A/G polymorphism is associated with Behçet's disease in a Tunisian population

The involvement of excessive T-helper cell functions in the pathogenesis of Behçet's disease (BD) has been reported. Cytotoxic T-lymphocyte antigen-4 (CTLA-4) plays a role in T-cell downregulation. In this report, we investigated the possible association between BD patients and the CTLA-4 +49A/G polymorphism in Tunisian population. A total of 135 Tunisian BD patients and 151 healthy blood donors from the same geographic area were genotyped by polymerase chain reaction for the CTLA-4 +49 A/G polymorphism. A highly significant difference between Tunisian BD patients and healthy controls was found regarding the distribution of CTLA-4 +49 A allele [ P  < 10⁻⁷; χ² = 75.63; odds ratio (OR) = 4.63; 95% confidence interval (CI) = 3.20–6.72] and genotype frequencies ( P  < 10⁻⁷; χ² = 71.02). Furthermore, in the BD group, the A allele was predominant in males (76.3%) when compared with females (62%), ( P  = 0.014; χ² = 5.97; OR = 1.99; 95% CI = 1.10–3.59). No relationship was found between the studied genotype and clinical manifestations. Our results show a gene dose effect of the A allele on the BD. The A allele exerts a stronger effect on disease susceptibility in males compared with females.     

Genetic polymorphism of esterase B3 in human leukocytes

Human tissues contain an esterase activity called ESB3, detectable by starch gel electrophoresis followed by staining with α-naphthyl butyrate. Using mononuclear leukocytes, we demonstrated an electrophoretic variant of ESB3. Family studies suggest that the variant is inherited as a simple Mendelian trait; individuals with the ESB3 2-1 phenotype are heterozygotes, designated ESB3 ¹ ESB3 ², to distinguish them from the more common homozygotes, ESB3 ¹ ESB3 ¹. The frequency of the ESB3 ² allele is estimated to be 0.035 in U.S. Whites. No homozygotes for this allele have yet been found.
Our studies suggest that the enzyme from ESB3 1 individuals exists primarily as a trimer of three identical subunits with a molecular weight of approximately 58000 daltons. The genetic variant ( ESB3 ² allele) appears to be the result of a mutation that does not affect the charge of the subunit, but rather reduces its ability to form and maintain the trimeric structure.     

The early history of the statistical estimation of linkage

The history of the statistical estimation of linkage is traced from its beginnings in 1912 to its coming-of-age in 1928, with special reference to the forgotten contribution of G. U. Yule and F. L. Engledow in 1914 introducing the method of minimum χ² as the first efficient procedure.