Double Heterozygotes Research Articles

Leg Ulcers in Sickle Cell Disease: Current Patterns and Practices

Leg ulcers are a debilitating complication of patients with sickle cell disease, and their frequency in North America was reported to be 2.5% by the Cooperative Study of Sickle Cell Disease more than 20 years ago. We sought to determine if the frequency of leg ulcers in sickle cell patients in the United States had declined and to assess which treatments providers use most commonly. We sent an e-mail survey to health professionals belonging to the national Sickle Cell Adult Provider Network. Responses were obtained from 31 of them (26.0%). Most of them (96.0%) reported having some patients with leg ulcers. Providers reported a total of 185 patients with active leg ulcers and 224 in the previous 5 years, for a total of 409 patients. Hb SS (homozygous sickle cell anemia) was the most common genotype of affected individuals, followed by Hb SC (double heterozygote for Hb S [β6(A3)Glu→Val, GAG>GTG; HBB: c.20A>T] and Hb C [β6(A3)Glu→Lys, GAG>AAG; HBB: c.19G>A]). Males showed a 2:1 predominance. Two-thirds of patients were treated with either hydroxyurea (HU) or transfusion therapy and most used compression stockings and topical therapies as directed by wound care services. We conclude that leg ulcers continue to be a debilitating complication of young adults with sickle cell disease, despite improved supportive care and the widespread use of disease modifying agents such HU and transfusion. While some providers offer office-based ulcer care, the majority prefer specialty consultation including podiatry, plastic surgery and dermatology. Despite their frequency, there is no clear consensus among providers as to the best treatment.

Doubly heterozygous LMNA and TTN mutations revealed by exome sequencing in a severe form of dilated cardiomyopathy

Familial dilated cardiomyopathy (DCM) is a heterogeneous disease; although 30 disease genes have been discovered, they explain only no more than half of all cases; in addition, the causes of intra-familial variability in DCM have remained largely unknown. In this study, we exploited the use of whole-exome sequencing (WES) to investigate the causes of clinical variability in an extended family with 14 affected subjects, four of whom showed particular severe manifestations of cardiomyopathy requiring heart transplantation in early adulthood. This analysis, followed by confirmative conventional sequencing, identified the mutation p.K219T in the lamin A/C gene in all 14 affected patients. An additional variant in the gene for titin, p.L4855F, was identified in the severely affected patients. The age for heart transplantation was substantially less for LMNA:p.K219T/TTN:p.L4855F double heterozygotes than that for LMNA:p.K219T single heterozygotes. Myocardial specimens of doubly heterozygote individuals showed increased nuclear length, sarcomeric disorganization, and myonuclear clustering compared with samples from single heterozygotes. In conclusion, our results show that WES can be used for the identification of causal and modifier variants in families with variable manifestations of DCM. In addition, they not only indicate that LMNA and TTN mutational status may be useful in this family for risk stratification in individuals at risk for DCM but also suggest titin as a modifier for DCM.

Spectrum of mutations and phenotypic expression in patients with autosomal dominant hypercholesterolemia identified in Italy

ObjectiveTo determine the spectrum of gene mutations and the genotype–phenotype correlations in patients with Autosomal Dominant Hypercholesterolemia (ADH) identified in Italy. MethodsThe resequencing of LDLR, PCSK9 genes and a selected region of APOB gene were conducted in 1018 index subjects clinically heterozygous ADH and in 52 patients clinically homozygous ADH. The analysis was also extended to 1008 family members of mutation positive subjects. ResultsMutations were detected in 832 individuals: 97.4% with LDLR mutations, 2.2% with APOB mutations and 0.36% with PCSK9 mutations. Among the patients with homozygous ADH, 51 were carriers of LDLR mutations and one was an LDLR/PCSK9 double heterozygote. We identified 237 LDLR mutations (45 not previously reported), 4 APOB and 3 PCSK9 mutations. The phenotypic characterization of 1769 LDLR mutation carriers (ADH-1) revealed that in both sexes independent predictors of the presence of tendon xanthomas were age, the quintiles of LDL cholesterol, the presence of coronary heart disease (CHD) and of receptor negative mutations. Independent predictors of CHD were male gender, age, the presence of arterial hypertension, smoking, tendon xanthomas, the scalar increase of LDL cholesterol and the scalar decrease of HDL cholesterol. We identified 13 LDLR mutation clusters, which allowed us to compare the phenotypic impact of different mutations. The LDL cholesterol raising potential of these mutations was found to vary over a wide range. ConclusionsThis study confirms the genetic and allelic heterogeneity of ADH and underscores that the variability in phenotypic expression of ADH-1 is greatly affected by the type of LDLR mutation.

Differential haplotype amplification leads to misgenotyping of heterozygote as homozygote when using single nucleotide mismatch primer

Mismatches at the 3'end of /or within a primer are reported to affect the efficiency of PCR and cause allele drop. Here, we report preferential amplification of one haplotype and misgenotyping, when double heterozygotes at NAT1 (rs1057126 and rs15561) were genotyped by sequencing and PCR-RFLP methods using mismatch reverse primers located next to the target SNP. Detailed study revealed highest (100%) and lowest (0%) misgenotyping when the mismatch was at the 3rd and 15th nucleotide positions from 3' end of the primer, respectively. But, the same primers, without any mismatch genotyped heterozygotes correctly. Homozygotes can always be detected correctly irrespective of mismatch position in the primer. Similar results were observed for two SNPs (rs12947788 and rs 12951053) at TP53. Using mismatch NAT1 reverse primers, located three nucleotides away from the target SNP, both TaqMan and sequencing methods showed preferential synthesis of one haplotype strand and misgenotyping in heterozygotes, respectively. So, mismatch primer, located next to target SNP, should be avoided to genotype heterozygotes, since, PCR and sequencing based genotyping methods may lead the investigators to report faulty allelic and genotypic frequencies. This study mimics a situation when an unknown variation is present in the primer-binding sites of both chromosomes.

Abstract 19819: Genetic Pre-Participation Screening in Selected Athletes: A New Tool for the Prevention of Sudden Cardiac Death?

Background: Sudden cardiac death (SCD) of athletes is a topical issue. Borderline cardiac abnormalities may result in SCD, which in turn may be genetically based; thus, genetic analysis may help to identify potentially pathological cardiac abnormalities. Aim: We performed phenotype-guided genetic analysis in athletes who, pre-participation, showed ECG and/or Echo borderline abnormalities, to discriminate subjects at a greater risk of SCD. Subjects and Methods: We studied 24 elite athletes referred by the National Federation of Olympic Sports; and 25 subjects seeking eligibility to practice agonistic sport referred by the Osservatorio Epidemiologico della Medicina dello Sport, Regione Campania. Inclusion criteria were: a) borderline ECG repolarization abnormalities; b) benign ventricular arrhythmias; c) LVWT in the grey zone of physiology versus pathology (MWT 12-15 mm in females; 13-16 mm in males). Based on the suspected phenotype, we screened subjects for LMNA gene, 8 sarcomeric genes, 5 desmosomal genes, and cardiac calcium, sodium and potassium channel disease genes. Results: Genetic analysis was completed in 37 out of 49 athletes, 22 competitive and 27 non-competitive athletes, showing borderline clinical markers suggestive of hypertrophic cardiomyopathy (HCM n 24), dilated cardiomyopathy (n 4), arrhythmogenic right ventricular dysplasia/catecholaminergic polymorphic ventricular tachycardia (ARVD/CPVT n 11), long QT syndrome (LQTS n 4), sick sinus syndrome (SSS n 5), or Brugada syndrome (BrS n 1). We identified 11 mutations in 9 athletes (an ARVD athlete was a compound heterozygote for the PKP2 gene and an HCM athlete was a double heterozygote for the MYBPC3 and TNNT2 genes): 3 known mutations related to LQTS, HCM and ARVD, respectively, and 8 novel mutations, located in the SCN5A, RyR2, PKP2, MYBPC3 and ACTC1 genes. The new mutations were absent from ∼800 normal chromosomes and were predicted probably damaging by in silico analysis. Patch clamp analysis in channelopathies indicated that some mutations were associated with abnormal biophysical behavior of the corresponding mutant protein. Conclusion: Genetic analysis may help distinguish between physiology and pathology in athletes with suspected familial or sporadic heart disease.

Mutation Analysis of BRCA1, BRCA2, PALB2 and BRD7 in a Hospital-Based Series of German Patients with Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) is an aggressive form of breast carcinoma with a poor prognosis. Recent evidence suggests that some patients with TNBC harbour germ-line mutations in DNA repair genes which may render their tumours susceptible to novel therapies such as treatment with PARP inhibitors. In the present study, we have investigated a hospital-based series of 40 German patients with TNBC for the presence of germ-line mutations in BRCA1, BRCA2, PALB2, and BRD7 genes. Microfluidic array PCR and next-generation sequencing was used for BRCA1 and BRCA2 analysis while conventional high-resolution melting and Sanger sequencing was applied to study the coding regions of PALB2 and BRD7, respectively. Truncating mutations in BRCA1 were found in six patients, and truncating mutations in BRCA2 and PALB2 were detected in one patient each, whereas no truncating mutation was identified in BRD7. One patient was a double heterozygote for the PALB2 mutation, c.758insT, and a BRCA1 mutation, c.927delA. Our results confirm in a hospital-based setting that a substantial proportion of German TNBC patients (17.5%) harbour germ-line mutations in genes involved in homology-directed DNA repair, with a preponderance of BRCA1 mutations. Triple-negative breast cancer should be considered as an additional criterion for future genetic counselling and diagnostic sequencing.

Homozygous factor V Leiden and double heterozygosity for factor V Leiden and prothrombin mutation

The most common forms of familial thrombophilia are factor V Leiden (FVL) and prothrombin mutation (PTM). Homozygous FVL and PTM have long been feared conditions thought to cause high rates of morbidity and mortality. To analyse clinical features in patients with homozygous FVL and PTM, as well as patients with double heterozygosity for FVL and PTM. All patients with homozygous FVL, PTM or double heterozygosity in the MATS database of 1465 consecutive unselected patients were analysed regarding age at inclusion venous thromboembolism (VTE), age at first thrombosis, recurrence, clinical course and acquired risk factors. We found 36 patients homozygous for FVL. Patients homozygous for FVL were younger than controls at group level (56±18 vs. 63±17, p<0.02). Homozygous women were younger than female controls (50±19 vs. 63±18, p<0.002). No difference was observed when comparing male subjects. Women were younger than men at inclusion thrombosis (50±19 vs. 65±14, p<0.02) and at first thrombosis (47±19 vs. 64±14, p<0.01). Deep venous thrombosis (DVT) was seen in 33 patients (92%), 6 (17%) had pulmonary embolism (PE) and 3 (8%) had combined DVT and PE. PE was less frequent in homozygous FVL women compared to female controls (p<0.03). VTE recurred in 3 subjects during the duration of the study. Odds ratio for VTE in homozygous FVL patients compared to controls was 13.9 (95% CI 9.9-19.7). We found no subjects with homozygous PTM. Double heterozygosity for FVL and PTM was seen in 12 subjects. There was no difference in age at inclusion VTE between double heterozygotes and controls (59±16 vs. 63±17, ns.). DVT was seen in 92% at inclusion, 8% had PE. Mean age at first VTE was 52±17 (27-82). Consecutive homozygous FVL patients had a higher age at first thrombosis than previously described. Homozygous females are affected at an earlier age than homozygous men and female controls. It seems that thrombi in homozygous FVL have a different pattern compared to controls i.e. more prone for thrombosis in the lower extremity. The odds ratio for thrombosis among homozygous FVL seems to be lower than previously described.

Selective Sweeps in Multilocus Models of Quantitative Traits

We study the trajectory of an allele that affects a polygenic trait selected toward a phenotypic optimum. Furthermore, conditioning on this trajectory we analyze the effect of the selected mutation on linked neutral variation. We examine the well-characterized two-locus two-allele model but we also provide results for diallelic models with up to eight loci. First, when the optimum phenotype is that of the double heterozygote in a two-locus model, and there is no dominance or epistasis of effects on the trait, the trajectories of selected mutations rarely reach fixation; instead, a polymorphic equilibrium at both loci is approached. Whether a polymorphic equilibrium is reached (rather than fixation at both loci) depends on the intensity of selection and the relative distances to the optimum of the homozygotes at each locus. Furthermore, if both loci have similar effects on the trait, fixation of an allele at a given locus is less likely when it starts at low frequency and the other locus is polymorphic (with alleles at intermediate frequencies). Weaker selection increases the probability of fixation of the studied allele, as the polymorphic equilibrium is less stable in this case. When we do not require the double heterozygote to be at the optimum we find that the polymorphic equilibrium is more difficult to reach, and fixation becomes more likely. Second, increasing the number of loci decreases the probability of fixation, because adaptation to the optimum is possible by various combinations of alleles. Summaries of the genealogy (height, total length, and imbalance) and of sequence polymorphism (number of polymorphisms, frequency spectrum, and haplotype structure) next to a selected locus depend on the frequency that the selected mutation approaches at equilibrium. We conclude that multilocus response to selection may in some cases prevent selective sweeps from being completed, as described in previous studies, but that conditions causing this to happen strongly depend on the genetic architecture of the trait, and that fixation of selected mutations is likely in many instances.

GnRH-Deficient Phenotypes in Humans and Mice With Heterozygous Variants in KISS1/Kiss1

Defects in a number of genes have been implicated in the pathophysiology of GnRH deficiency. Several lines of evidence suggest that KISS1 is a likely candidate gene for GnRH deficiency. These include identification of inactivating mutations in KISS1R (a.k.a. GPR54) in patients with GnRH deficiency, targeted deletions of either KISS1 or KISS1R in mice with hypogonadotropic phenotypes, and the demonstrated role of kisspeptin in stimulation of GnRH neurons and initiation of puberty. However, no inactivating mutations in KISS1 have been found in patients with GnRH deficiency. The aim of this study was to identify deleterious mutations in KISS1. After a systematic evaluation of genetic variation identified rare sequence variants (RSV) in KISS1 among 1025 probands with GnRH deficiency, the frequency and functional significance of RSV were studied. DNA sequencing was performed in RSV of all probands and controls with normal reproductive function (n = 535). In addition to human studies, reproductive phenotypes in heterozygous and double-heterozygous KISS1 and KISS1R mice were examined to test the hypothesis that heterozygous mutations in KISS1 can cause reproductive deficits either alone or in combination with mutations in other genes. Individual variants located within the mature kisspeptin peptide were added to CHO-KISS1R cells to measure the accumulation of inositol phosphate. Variants located outside the peptide coding region were also analyzed in vitro and in silico. Transcriptional efficiency of the g.1→3659C3T variant was examined in vitro. Among the 1025 probands, 15 carried heterozygous RSV in KISS1 seen in less than 1% of the controls. Of 5 variants located within the mature kisspeptin peptide, p.F117L (but not p.S77I, p.Q82K, p.H90D, or p.P110T) reduced inositol phosphate generation in vitro. Analysis in silico showed that 2 variants residing within the coding region but outside the mature peptide, p.G35S and p.C53R (but not p.A129V), were predicted to be inactivating. One variant residing outside the coding region (g.1→3659C3T impairs transcription in vitro and another (c.1→7C3T) lies within the consensus Kozak sequence. Four of 5 probands tested had abnormal endogenous LH pulse patterns at baseline. In the mouse model, serum testosterone concentration decreased with increasing heterozygous loss of Kiss1 and Kiss1r alleles [wild-type, 274 (99) vs double heterozygotes, 69 (16) ng/dL; r2 = 0.13; P = 0.03]. Compared with wild-type mice, Kiss1/Kiss1r double-heterozygote males had shorter anogenital distances [13.0 (0.2) vs 15.6 (0.2) mm at P34, P < 0.001] and females had longer estrous cycles [7.4 (0.2) vs 5.6 (0.2) days, P < 0.01]. In addition, mating pairs had decreased litter frequency [0.59 (0.09) vs 0.71 (0.06) litters/month, P < 0.04] and size [3.5 (0.2) vs 5.4 (0.3) pups/litter, P < 0.001]. These findings show that rare, deleterious, heterozygous variants in KISS1, which seem to be associated with GnRH deficiency and abnormal reproductive phenotypes in humans, exist. GnRH deficiency in humans is not caused by KISS1 RSV alone. As in the mouse model, it may result from heterozygous KISS1 variants acting in concert with other genetic factors and/or environmental factors.

Identification of an RP1 Prevalent Founder Mutation and Related Phenotype in Spanish Patients with Early-Onset Autosomal Recessive Retinitis

To identify the genetic causes underlying early-onset autosomal recessive retinitis pigmentosa (arRP) in the Spanish population and describe the associated phenotype. Case series. A total of 244 unrelated families affected by early-onset arRP. Homozygosity mapping or exome sequencing analysis was performed in 3 families segregating arRP. A mutational screening was performed in 241 additional unrelated families for the p.Ser452Stop mutation. Haplotype analysis also was conducted. Individuals who were homozygotes, double heterozygotes, or carriers of mutations in RP1 underwent an ophthalmic evaluation to establish a genotype-phenotype correlation. DNA sequence variants, homozygous regions, haplotypes, best-corrected visual acuity, visual field assessments, electroretinogram responses, and optical coherence tomography images. Four novel mutations in RP1 were identified. The new mutation p.Ser542Stop was present in 11 of 244 (4.5%) of the studied families. All chromosomes harboring this mutation shared the same haplotype. All patients presented a common phenotype with an early age of onset and a prompt macular degeneration, whereas the heterozygote carriers did not show any signs of retinitis pigmentosa (RP). p.Ser542Stop is a single founder mutation and the most prevalent described mutation in the Spanish population. It causes early-onset RP with a rapid macular degeneration and is responsible for 4.5% of all cases. Our data suggest that the implication of RP1 in arRP may be underestimated. The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Multiple jejunal cancers resulting from combination of germline APC and MLH1 mutations

Double heterozygotes for mutations in APC and a DNA mismatch repair gene are extremely rare. We report on an individual who had truncating mutations in APC and MLH1 whose clinical presentation initially resembled Familial Adenomatous Polyposis but then emerged as a novel phenotype with multiple jejunal carcinomas. We have reviewed the relevant literature on double heterozygotes and based on what has been reported to date, this phenotype was not anticipated. It may be useful for clinicians to be aware of this observation as clinical screening guidelines are proposed for such individuals.

The Wnt Receptor Ryk Plays a Role in Mammalian Planar Cell Polarity Signaling

Wnts are essential for a wide range of developmental processes, including cell growth, division, and differentiation. Some of these processes signal via the planar cell polarity (PCP) pathway, which is a β-catenin-independent Wnt signaling pathway. Previous studies have shown that Ryk, a member of the receptor tyrosine kinase family, can bind to Wnts. Ryk is required for normal axon guidance and neuronal differentiation during development. Here, we demonstrate that mammalian Ryk interacts with the Wnt/PCP pathway. In vitro analysis showed that the Wnt inhibitory factor domain of Ryk was necessary for Wnt binding. Detailed analysis of two vertebrate model organisms showed Ryk phenotypes consistent with PCP signaling. In zebrafish, gene knockdown using morpholinos revealed a genetic interaction between Ryk and Wnt11 during the PCP pathway-regulated process of embryo convergent extension. Ryk-deficient mouse embryos displayed disrupted polarity of stereociliary hair cells in the cochlea, a characteristic of disturbed PCP signaling. This PCP defect was also observed in mouse embryos that were double heterozygotes for Ryk and Looptail (containing a mutation in the core Wnt/PCP pathway gene Vangl2) but not in either of the single heterozygotes, suggesting a genetic interaction between Ryk and Vangl2. Co-immunoprecipitation studies demonstrated that RYK and VANGL2 proteins form a complex, whereas RYK also activated RhoA, a downstream effector of PCP signaling. Overall, our data suggest an important role for Ryk in Wnt/planar cell polarity signaling during vertebrate development via the Vangl2 signaling pathway, as demonstrated in the mouse cochlea.

The Limits to Parapatric Speciation: Dobzhansky–Muller Incompatibilities in a Continent–Island Model

How much gene flow is needed to inhibit speciation by the accumulation of Dobzhansky-Muller incompatibilities (DMIs) in a structured population? Here, we derive these limits in a classical migration-selection model with two haploid or diploid loci and unidirectional gene flow from a continent to an island. We discuss the dependence of the maximum gene-flow rate on ecological factors (exogeneous selection), genetic factors (epistasis, recombination), and the evolutionary history. Extensive analytical and numerical results show the following: (1) The maximum rate of gene flow is limited by exogeneous selection. In particular, maintenance of neutral DMIs is impossible with gene flow. (2) There are two distinct mechanisms that drive DMI evolution in parapatry, selection against immigrants in a heterogeneous environment and selection against hybrids due to the incompatibility. (3) Depending on the mechanism, opposite predictions result concerning the genetic architecture that maximizes the rate of gene flow a DMI can sustain. Selection against immigrants favors evolution of tightly linked DMIs of arbitrary strength, whereas selection against hybrids promotes the evolution of strong unlinked DMIs. In diploids, the fitness of the double heterozygotes is the decisive factor to predict the pattern of DMI stability.

Igf2 pathway dependency of the Trp53 developmental and tumour phenotypes.

Insulin-like growth factor 2 (IGF2) and the transformation related protein 53 (Trp53) are potent regulators of cell growth and metabolism in development and cancer. In vitro evidence suggests several mechanistic pathway interactions. Here, we tested whether loss of function of p53 leads to IGF2 ligand pathway dependency in vivo. Developmental lethality occurred in p53 homozygote null mice that lacked the paternal expressed allele of imprinted Igf2. Further lethality due to post-natal lung haemorrhage occurred in female progeny with Igf2 paternal null allele only if derived from double heterozygote null fathers, and was associated with a specific gene expression signature. Conditional deletion of Igf2fl/fl attenuated the rapid tumour onset promoted by homozygous deletion of p53fl/fl. Accelerated carcinoma and sarcoma tumour formation in p53+/− females with bi-allelic Igf2 expression was associated with reductions in p53 loss of heterozygosity and apoptosis. Igf2 genetic dependency of the p53 null phenotype during development and tumour formation suggests that targeting the IGF2 pathway may be useful in the prevention and treatment of human tumours with a disrupted Trp53 pathway.

Acute impact of apheresis on oxidized phospholipids in patients with familial hypercholesterolemia

We measured oxidized phospholipids (OxPL), lipoprotein (a) [Lp(a)], and lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) pre- and postapheresis in 18 patients with familial hypercholesterolemia (FH) and with low(∼10 mg/dl; range 10-11 mg/dl), intermediate (∼50 mg/dl; range 30-61 mg/dl), or high (>100 mg/dl; range 78-128 mg/dl) Lp(a) levels. By using enzymatic and immunoassays, the content of OxPL and Lp-PLA(2) mass and activity were quantitated in lipoprotein density fractions plated in microtiter wells, as well as directly on apoB-100, Lp(a), and apoA-I immunocaptured within each fraction (i.e., OxPL/apoB and Lp-PLA(2)/apoB). In whole fractions, OxPL was primarily detected in the Lp(a)-containing fractions, whereas Lp-PLA(2) was primarily detected in the small, dense LDL and light Lp(a) range. In lipoprotein capture assays, OxPL/apoB and OxPL/apo(a) increased proportionally with increasing Lp(a) levels. Lp-PLA(2)/apoB and Lp-PLA(2)/apoA-I levels were highest in the low Lp(a) group but decreased proportionally with increasing Lp(a) levels. Lp-PLA(2)/apo(a) was lowest in patients with low Lp(a) levels and increased proportionally with increasing Lp(a) levels. Apheresis significantly reduced levels of OxPL and Lp-PLA(2) on apoB and Lp(a) (50-75%), particularly in patients with intermediate and high Lp(a) levels. In contrast, apheresis increased Lp-PLA(2)-specific activity (activity/mass ratio) in buoyant LDL fractions. The impact of apheresis on Lp(a), OxPL, and Lp-PLA(2) provides insights into its therapeutic benefits beyond lowering apoB-containing lipoproteins.

Study on correlation between erythrocyte indices and genotype in cases with alpha-thalassemia

Objective To investigate the correlation between the erythrocyte indices and the genotypes of alpha thalassemia. Methods 337 carriers with various genotypes of alpha-thalassaemia (iron deficiency, alpha-thalassemia double heterozygote and homozygote, α-compounding β-thalassemia and abnormal hemoglobinopathy were excluded) were classified into three groups based on different genotypes of alpha-thalassaemia including silent thalassemia group (ST,83 cases), α-thalassemia trait group (TT,210 cases) and intermediate thalassemia group（IT,44 cases）,and 154 healthy adults were randomly choosed as normal control.The erythrocyte indices involving in RBC,Hb,MCV, MCH, MCHC and RDW-CV were retrospectively analyzed and the difference of which was compared by analysis of variance and SNK test among aboved-mentioned groups. Results There were statistical significance among groups about erythrocyte indices except Hb F.The order of the level of MCV and MCH was NC［（86.6±5.2） fl,(29.5±2.1） pg］＞ST［（80.1±3.3） fl,(26.7±1.3） pg］＞TT［（68.5±3.4） fl,(22.0±1.2) pg ］＞IT［（66.6±7.1） fl,(20.0±2.2) pg，F=580.67,761.19，P<0.05］.And the size of RDW-CV was IT（22.3±3.4）%＞TT(14.9±1.2) %＞ST（13.8±1.6）%＞NC（13.2±1.4）%（F=347.25，P<0.05）.In ST group, the value of MCHC of -α3.7/αα subgroup（335.6±8.0） g/L was higher than that of -α4.2/αα subgroup（330±7.2） g/L and αTα/αα subgroup （328.4±9.5） g/L（F=6.07，P<0.05）.Meanwhile, in IT group, the value of MCV of αTα/--SEA subgroup（70.1±7.2） fl was higher than that of -α3.7/--SEA subgroup (63.4±5.9) fl and -α4.2/--SEA subgroup (64.1±4.0) fl（F=5.55，P<0.05）.However, the value of MCHC of αTα/--SEA subgroup（289.7±21.2） g/L was lower than that of other two subgroups［(306.3±8.4), (306.1±8.7) g/L，F=8.72，P<0.05］.Except Hb A2 and Hb F, there was positive correlation between the number of deleted α-globin gene and that of RBC and RDW-CV（r=0.318 and 0.580，P<0.01）.Nevertheless, there was negative correlation between the number of deleted α-globin gene and that of the other erythrocyte indices (r= -0.483, -0.827, -0.744 and -0.684, P all<0.01）. Conclusions There is close correlation between the degree of anemia and the number of deleted α-globin gene characterized by Hb reduction and RBC increasing.In addition, the anemia degree of non-deletional Hb H disease is severer than that of deletional Hb H,which of Hb H disease with -α4.2/--SEA is severer than that with -α3.7/--SEA.(Chin J Lab Med, 2012，35：418-422) Key words: alpha-Thalassemia; Erythrocyte indices; Genotype

073 Evaluation of clinical markers of early disease expression and the ability to predict genotype in families with HCM and mutations in cardiac myosin binding protein C

IntroductionFamilial evaluation for the presence of left ventricular hypertrophy (LVH) is an important part of management in hypertrophic cardiomyopathy. However due to incomplete penetrance, the presence of LVH does not...

Maximum Likelihood Estimation of Linkage Disequilibrium in Half-Sib Families

Maximum likelihood methods for the estimation of linkage disequilibrium between biallelic DNA-markers in half-sib families (half-sib method) are developed for single and multifamily situations. Monte Carlo computer simulations were carried out for a variety of scenarios regarding sire genotypes, linkage disequilibrium, recombination fraction, family size, and number of families. A double heterozygote sire was simulated with recombination fraction of 0.00, linkage disequilibrium among dams of δ=0.10, and alleles at both markers segregating at intermediate frequencies for a family size of 500. The average estimates of δ were 0.17, 0.25, and 0.10 for Excoffier and Slatkin (1995), maternal informative haplotypes, and the half-sib method, respectively. A multifamily EM algorithm was tested at intermediate frequencies by computer simulation. The range of the absolute difference between estimated and simulated δ was between 0.000 and 0.008. A cattle half-sib family was genotyped with the Illumina 50K BeadChip. There were 314,730 SNP pairs for which the sire was a homo-heterozygote with average estimates of r2 of 0.115, 0.067, and 0.111 for half-sib, Excoffier and Slatkin (1995), and maternal informative haplotypes methods, respectively. There were 208,872 SNP pairs for which the sire was double heterozygote with average estimates of r2 across the genome of 0.100, 0.267, and 0.925 for half-sib, Excoffier and Slatkin (1995), and maternal informative haplotypes methods, respectively. Genome analyses for all possible sire genotypes with 829,042 tests showed that ignoring half-sib family structure leads to upward biased estimates of linkage disequilibrium. Published inferences on population structure and evolution of cattle should be revisited after accommodating existing half-sib family structure in the estimation of linkage disequilibrium.

Abstract 134: A Double Heterozygote for LCAT and ABCA1 Mutations Accounts for Severe Hypoalphalipoproteinemia

We report a family with extremely depressed high density lipoprotein (HDL) cholesterol (C) levels. The proband, a 27 year-old male of Asian Indian origin, was identified on routine lipid screening to have an HDL-C of 9mg/dL. His 24 year-old brother had an HDL-C of 6mg/dL and was therefore referred to a local lipid clinic in 1997. Both brothers were asymptomatic. The nuclear family was comprised of another brother and a mother living in India; their father had died previously from an undetermined cause. The parents were second cousins. Blood was obtained from members of the extended family and showed that the mother’s HDL-C was reduced at 29mg/dL, while the 3rd bother had a normal HDL-C at 54mg/dL. Based on the above scenario, a homozygous mutation most likely involving ABCA1 was presumed to be responsible for this lipoprotein profile. However, microsatellite marker analyses of the ABCA1 locus showed no homozygosity pattern in the two affected brothers. Further analyses of the APOA1-C3-A4 locus similarly failed to show homozygosity in the affected brothers. Plasma activities of both salt-stimulated paraoxanase and LCAT were at the low end of normal. Direct sequence analyses of the LCAT gene showed all three brothers were heterozygous for the Arg423His mutation, while the mother’s LCAT gene sequence was normal. No further work was pursued until 2011, when this family’s DNA was re-examined by direct sequencing. On this occasion, a heterozygous mutation in ABCA1 was found, namely Arg1270X in the 2 affected brothers and their mother. To our knowledge, this is the first report of ’double heterozygosity’ for LCAT and ABCA1 mutations producing severe hypoalphalipoproteinemia. Further, simple heterozygosity for the ABCA1 Arg1270X mutation had a co-dominant influence, moderately reducing HDL-C levels. In contrast, simple heterozygosity for the LCAT Arg423His mutation had no effect on HDL-C, but in the presence of ABCA1 Arg1270X it contributed to markedly reduced HDL-C. This case report highlights a potential pitfall in human genetic studies, namely assuming a particular inheritance pattern based upon family structure. It also highlights the value of perseverance by searching for causative mutations in more than one gene.

Genetic Polymorphism of γ-Glutamyl Hydrolase in Chinese Acute Leukemia Children and Identification of a Novel Double Nonsynonymous Mutation

γ-Glutamyl hydrolase (GGH) plays a central role in folate metabolism and antifolate action. Polymorphism in the human GGH gene (GGH) was associated with efficacy and side effects of methotrexate for treatment of acute lymphoblastic leukemia (ALL). This study aimed to identify polymorphisms of GGH in Chinese. Seventy-one children with ALL, 25 children with acute myeloid leukemia (AML), and 132 children with nonmalignancy as control were included. Human GGH cDNAs were prepared and analyzed by reverse transcriptase–polymerase chain reaction (RT-PCR)–denaturing gradient gel electrophoresis. The detected variants were further confirmed by DNA sequencing. Novel variant GGH cDNA was cloned, expressed, and characterized. Allelic frequency of GGH 452C→T polymorphism was determined as 9.2% (CT, 11/71; TT, 1/71) in the ALL group, 8.0% (CT, 4/25; TT, 0/25) in the AML group, and 9.1% (TT, 4/132; T/C, 16/132) in the controls, respectively. The total allelic frequency in the Chinese population (9.0%) was higher than those reported in Japanese (5.6%) and African Americans (4.4%), and was similar to Caucasians (10.0%). Association of 452TT+TC with increased rate of hepatotoxicity and mucositis was observed in the ALL patients. Two novel mutations were determined in the coding region of GGH in 2 boys with ALL: one of the mutations was a double nonsynonymous heterozygote (841AG+845AG, K257E/N258S), the other was a nonsynonymous heterozygote (797AG, K242R). The corresponding double-mutant protein showed unchanged enzymatic activity by functional analysis. Allele frequency of GGH C452T polymorphism is determined for the first time among Chinese. A novel double nonsynonymous mutation of GGH was identified in a boy with ALL.