HLA and trisomy 21. Confirmation of a trend of restricted HLA heterogeneity in parents of Down syndrome children

As the HLA system could play a role in the in utero selection process against abnormal fetuses, HLA-A and -B antigens were evidenced in 30 children with trisomy 21 and in their parents, using a standard microlymphocytotoxicity test. The comparison group included 60 families among whom 39 had HLA typing for paternity exclusion and 21 had been previously selected for a segregation study. Both groups consisted of nonconsanguineous Caucasians from the same geographical area. The Down syndrome (DS) children did not show a significant association with a specific HLA antigen. However, six out of 30 couples having a DS child showed two antigens shared at the A and/or B locus, compared to seven out of 60 control couples. The shared parental antigens were not selectively inherited, and the proportion of homozygote children at one locus was lower for DS (5/30) than for controls (13/60). These findings demonstrate the same trend as previously published but need to be confirmed by other investigators. Perhaps a strong selective pressure in favor of heterozygotes contributes to a better survival rate, as suggested from histocompatibility studies in animals.     

Parental HLA compatibility, fetal wastage and neural tube defects: evidence for a T/t-like locus in humans

To test the hypothesis that a locus in or near the human major histocompatibility complex (HLA) contributes to both involuntary fetal loss and neural tube defects (NTD), we evaluated sharing of antigens of the HLA-A, HLA-B, or HLA-DR loci of couples who had three or more first-trimester spontaneous abortions or who had a child with an NTD (myelomeningocele or anencephaly). HLA-A antigen sharing was increased in couples with three or more spontaneous abortions and in couples who had an anencephalic fetus, when compared with couples who had three or more pregnancies and no fetal loss. Increased sharing of antigens at the HLA-A and B loci was not seen in the entire group of couples with children with myelomeningocele, but was found in the subgroup of 36 couples whose child had a lumbar myelomeningocele. An increase in HLA-DR sharing was not seen in any group or subgroup when compared with the control couples. Among the aborting couples, increased sharing was not restricted to the couples who had no term pregnancies, but was also found in the couples whose fetal losses occurred after one or more normal term pregnancies. These results are consistent with the hypothesis that a locus on the HLA-A side of the HLA-DR locus contributes to some fetal loss and susceptibility to NTD. This model is proposed as an alternative to the hypothesis that the maternal immune response to paternal major histocompatibility complex (MHC) antigens is the basis for increased HLA sharing in couples with fetal wastage.     

Grandmaternal age in children with Down syndrome in St. Petersburg

Advanced maternal age is a well-established factor of DS occurrence. However the majority of DS cases are born to young couples. Some studies suggested that the risk for Down syndrome may be related to an aging grandmother. We obtained data on grandmaternal ages in 243 families of DS and 330 families of healthy children born in 1990-1999. The data were analyzed according to two categories of maternal ages, <30 yr and > or =30 yr. We did not find systematic differences in grandparental age distribution between the studied groups. Specifically, in 102 young couples with DS, medians for both maternal and paternal grandmother's age appeared to be equal (26 yr). Similar figures were observed in 284 young controls (27 yr). There was no difference in age distribution between 141 older couples with DS and 104 control couples. Therefore we failed to support the suggestion that advanced age of the DS grandmother is responsible for meiotic disturbance in her daughter. Neither the hypothesis suggesting a significant contribution of parentally transmitted trisomy 21 to DS population rate has been confirmed.     

Shared HLA antigens and reproductive performance among Hutterites.

Shared histocompatibility antigens between spouses may affect reproductive outcome adversely as a result of prenatal selection against compatible fetuses. Evidence from both animal and human studies suggest that histocompatible fetuses may not initiate a maternal immunologic response that prevents rejection of the embryo. Therefore, parents sharing HLA antigens may produce compatible fetuses and consequently experience a greater frequency of early fetal losses and show poorer reproductive outcome than couples not sharing antigens. In the Hutterites, an inbred human isolate that proscribes contraception, we tested the hypothesis that couples sharing HLA antigens have poorer reproductive outcomes than couples who do not. The Hutterites are characterized by high fertility and large family sizes. Couples that share zero (no. = 21), one (no. = 15), and more than one (no. = 10) HLA-A or HLA-B antigens were compared for reproductive performance. Median intervals between births were larger among couples that share more than one antigen in eight of 11 intervals examined. In addition, the median intervals from marriage to first, fifth, and tenth birth were consistently larger among couples that share more than one antigen. Differences among the groups appear to become larger with increasing parity, suggesting that the effect of histocompatibility on reproductive performance becomes more evident in later pregnancies. These differences in reproductive performance between couples that share zero, one, or more than one HLA-A or HLA-B antigens may have significant evolutionary consequences. However, our results demonstrate that sharing HLA antigens does not preclude normal pregnancy and caution should be exercised before concluding that shared HLA antigens are solely responsible for repeated fetal losses.     

Neutrophil alkaline phosphatase in children with trisomy 21 and their parents

A comparative study of karyotypes and neutrophil alkaline phosphatase (NAP) was carried out for 70 parents (35 couples) of trisomy 21 children and their 35 trisomy 21 children and for 110 control parents (55 couples) and their normal children. In the trisomy 21 families we found a significant increase in NAP: mother P less than 10-4; father P less than 10-4; children P less than 10-9; the NAP level in affected child is approximately equal to the sum of the NAP levels of the two parents (P = 0.80; sigma2 = 5%). In one parent of a trisomy 21 child, a karyotype anomaly was present.     

Reproductive failure and the major histocompatibility complex.

The association between HLA sharing and recurrent spontaneous abortion (RSA) was tested in 123 couples and the association between HLA sharing, and the outcome of treatment for unexplained infertility by in vitro fertilization (IVF) was tested in 76 couples, by using a new shared-allele test in order to identify more precisely the region of the major histocompatibility complex (MHC) influencing these reproductive defects. The shared-allele test circumvents the problem of rare alleles at HLA loci and at the same time provides a substantial gain in power over the simple chi 2 test. Two statistical methods, a corrected homogeneity test and a bootstrap approach, were developed to compare the allele frequencies at each of the HLA-A, HLA-B, HLA-DR, and HLA-DQ loci; they were not statistically different among the three patient groups and the control group. There was a significant excess of HLA-DR sharing in couples with RSA and a significant excess of HLA-DQ sharing in couples with unexplained infertility who failed treatment by IVF. These findings indicate that genes located in different parts of the class II region of the MHC affect different aspects of reproduction and strongly suggest that the sharing of HLA antigens per se is not the mechanism involved in the reproductive defects. The segment of the MHC that has genes affecting reproduction also has genes associated with different autoimmune diseases, and this juxtaposition may explain the association between reproductive defects and autoimmune diseases.     

The role of cytologic NOR variants in the etiology of trisomy 21.

Silver-stained chromosomes from 29 couples with a trisomy 21 offspring and from 25 control couples were studied to determine whether there was an association of nucleolar-organizing-region variants in parents of children with trisomy 21. A reproducible scoring system for the analysis of silver-stained chromosomes was developed, and this was applied to the analysis of study participants in a blinded fashion. Seven of the 58 parents of children with trisomy 21 and seven of the 50 control parents were found to have variant NORs on silver staining. Therefore, we do not find a demonstrable risk for nondisjunction of chromosome 21 in individuals with silver-staining variants.     

Cytochemical and biochemical studies on neutrophil alkaline phosphatase in parents of trisomy 21 children

Summary A study of karyotypes and neutrophil alkaline phosphatase (NAP) was carried out for 66 parents (33 couples) of trisomy 21 children and for 60 control parents (30 couples). Enzyme activity was determined simultaneously by biochemical and cytochemical techniques. In the mothers of trisomy 21 children we found: (a) by biochemical techniques, a significant increase of NAP activity in polymorphonuclear leukocyte (PMN) homogenates (P<0.01) and a lower supernatant/pellet ratio (P<0.01); (b) by cytochemical techniques (with or without thermal and urea treatments) NAP activity was significantly higher (P<0.001) than in control mothers; in the fathers, the two techniques gave normal NAP activity results.     

Grandmaternal ages at birth of parents of children with down syndrome in St. Petersburg

Data on the age of grandparents of 243 children with Down syndrome (DS) born between 1990 and 1999 are considered in this work in comparison with control families of 330 healthy children. In 102 families, where the age of the mother at the birth of a child with DS was younger 30 (or less than 30) years, the median ages of both maternal and paternal grandmothers of probands were actually the same (26 years). Actually, the median age of grandmothers in 226 young families having healthy children were also the same (27 years). No differences in the indicators in question were revealed between 141 families with DS and 104 families with healthy children, where the mothers were older than 29 years. Thus, our results have not confirmed the hypothesis about the influence of the age of DS probands’ grandmothers on the segregation of chromosomes in their daughters’ oogenesis, as well as the hypothesis about a significant contribution of the inherited trisomy of chromosome 21 to the frequency of DS in the general population.     

Homocysteine metabolism in children with Down syndrome: in vitro modulation

The gene for cystathionine beta-synthase (CBS) is located on chromosome 21 and is overexpressed in children with Down syndrome (DS), or trisomy 21. The dual purpose of the present study was to evaluate the impact of overexpression of the CBS gene on homocysteine metabolism in children with DS and to determine whether the supplementation of trisomy 21 lymphoblasts in vitro with selected nutrients would shift the genetically induced metabolic imbalance. Plasma samples were obtained from 42 children with karyotypically confirmed full trisomy 21 and from 36 normal siblings (mean age 7.4 years). Metabolites involved in homocysteine metabolism were measured and compared to those of normal siblings used as controls. Lymphocyte DNA methylation status was determined as a functional endpoint. The results indicated that plasma levels of homocysteine, methionine, S-adenosylhomocysteine, and S-adenosylmethionine were all significantly decreased in children with DS and that their lymphocyte DNA was hypermethylated relative to that in normal siblings. Plasma levels of cystathionine and cysteine were significantly increased, consistent with an increase in CBS activity. Plasma glutathione levels were significantly reduced in the children with DS and may reflect an increase in oxidative stress due to the overexpression of the superoxide dismutase gene, also located on chromosome 21. The addition of methionine, folinic acid, methyl-B(12), thymidine, or dimethylglycine to the cultured trisomy 21 lymphoblastoid cells improved the metabolic profile in vitro. The increased activity of CBS in children with DS significantly alters homocysteine metabolism such that the folate-dependent resynthesis of methionine is compromised. The decreased availability of homocysteine promotes the well-established "folate trap," creating a functional folate deficiency that may contribute to the metabolic pathology of this complex genetic disorder.     

Participation of Indo-European tribes in ethnogeny of the mongoloid population of Siberia: analysis of the HLA antigen distribution in mongoloids of Siberia.

Three hundred forty-three Yakuts (mongoloids of central Asian type living in Siberia) were tested for HLA-A, -B, and -C loci. The HLA antigen distribution corresponds on the whole to a mongoloid population with high frequency of the HLA-A9, -B15, and -B40 antigens (phenotype frequencies .533, .367, and .405, respectively). At the same time a strikingly high frequency for the "Indo-European" HLA-A1 antigen (phenotype frequency .282) was detected, which in Yakuts is found exclusively with HLA-B17 (haplotype frequency x 1,000 = 87.0; linkage disequilibrium value x 1,000 = 63.8). The present paper deals with a new hypothesis of the Yakut ethnogenesis according to which ancient Aryan tribes formed the substratum which was later assimilated by the mongoloid and Turkic populations. Another hypothesis that I have advanced argues that from analysis of the HLA system the ancient Aryans formed, a local group within the Indo-European entity, with high frequency for HLA-A1 and -B17 antigens and for the HLA-A1,B17 haplotype and with a complete absence of or very low frequency for the HLA-B8 antigen and for the HLA-A1,B8 haplotype. Significant linkage disequilibrium, as it is found in Indians and Yakuts, etc., could have resulted from mixing of the Aryans with non-Indo-European tribes. No significant linkage disequilibrium between A1 and B8 characteristic of the European caucasoids was produced in the mixing.     

Detection of mosaicism in lymphocytes of parents of free trisomy 21 offspring

Down syndrome (DS) resulting from free trisomy 21 (FT21) has been largely associated with advanced maternal age. However, approximately 60% of FT21 cases are born to young couples. Thus, the etiological factors responsible for these FT21 children must differ from those proposed for maternal age-related FT21. These factors have not been defined. In this study, we analyzed the chromosomes of peripheral blood lymphocytes from three groups of couples aged ≤35 years, to identify chromosomal trisomies: Group I included 5 couples with normal offspring; Group II included 22 couples with one FT21 child; and Group III consisted of 3 couples with recurrent FT21. A total of 13,809 metaphases were analyzed with G-banding and 60,205 metaphases were analyzed with FISH using a 13/21 centromeric probe. Aneuploidy was significantly more frequent in Groups II and III. The frequencies of hyperdiploid cells were 0.19, 0.49 and 0.96% in Groups I–III, respectively. FISH analysis showed that trisomy 21 cell percentages were 0.08, 0.21 and 0.76 for Groups I–III, respectively, and were very similar to those obtained with G-banding. Trisomy 21 mosaicism was found in 2/22 couples with one FT21 offspring, and in 2/3 couples with recurrent FT21. Our data suggest that mosaicism is an important cause of FT21 offspring in young couples, and that aneuploidy is more frequent among couples with FT21 offspring. This may be related with age and other undetermined intrinsic and extrinsic factors.     

Heterotrisomy, a significant contributing factor to ventricular septal defect associated with Down syndrome?

Down syndrome (DS; trisomy 21) is associated with a wide range of variable clinical features, one of the most common being congenital heart defects (CHD). We used molecular genetic techniques to study the inheritance of genes on chromosome 21 in children with DS and CHD. Polymorphic markers on the long arm of chromosome 21 were analysed in 99 families who had a child with DS. Of these, 60 children had a CHD and 39 children had no CHD. Heterotrisomy describes the inheritance of an allele from each of three different grandparents. In some cases heterotrisomy will involve the inheritance of three different alleles. Heterotrisomic regions were defined as those showing retention of non-disjoining parental heterozygosity at polymorphic loci in the non-disjoined chromosomes of children with DS. Using polymorphic non-coding markers, we identified a consistent 9.6-cM minimum region (D21S167-HMG14) of heterotrisomy in children with DS and ventricular septal defect (VSD). Comparing individuals with DS and VSD to all others with DS (those either with no CHD or with any other CHD combined) shows the individuals with DS and VSD to have significantly more non-reduction or heterotrisomy in this region (P=0.006, Fisher's exact test, two-tailed). We postulate that heterotrisomy for a gene or genes in this region is a contributing factor to the pathogenesis of VSD in trisomy 21 either through the presence of three different specific alleles or through the presence of specific combinations of alleles.     

Quantification of soluble HLA class I gene products by an enzyme linked immunosorbent assay

A simplified enzyme linked immunosorbent assay utilizing an HLA class I framework-specific monoclonal antibody and a polyclonal enzyme linked beta-2 microglobulin specific antiserum has been established for the quantitative measurement of soluble HLA class I molecules. A total of 219 unrelated healthy individuals and 137 members of 28 families typed for HLA were analyzed for their non-membrane bound, i.e. soluble HLA-A,B,C antigens (sHLA-A,B,C). As reported by others, we observed associations of higher or lower sHLA-A,B,C values to particular HLA antigens: High plasma values were observed in probands positive for HLA-A23, A24, A29, Aw33, Bw65, and Cw8 and low values in HLA-B27 and B37 positive individuals. However, as shown by family studies, levels of sHLA-A,B,C were apparently not controlled by the MHC haplotypes alone, since no significant difference between HLA identical siblings and two haplotype different individuals could be detected. Thus, additional non-MHC linked gene(s) may be involved in the release of class I gene products.     

HLA-associated susceptibility to acquired immune deficiency syndrome in HIV-1-seropositive subjects

No difference in HLA-A, B or DR gene frequencies could be observed between 172 control subjects and 180 HIV-1-seropositive subjects of European ancestry diagnosed through the systematic screening of blood donations. In contrast, progression to acquired immune deficiency syndrome (AIDS; 21 patients) or CD4 lymphocyte loss equal or more than 20% over a 6-month period (37 subjects) was found to be associated with the B8DR3 haplotype (relative risk = 10.64, p less than 0.003, and 2.23, p less than 0.092, respectively). Other independently significant associations assessed through the multivariate Cox proportional-hazards model were B16, BW21 and B35 alleles as factors of bad prognosis. Conversely, A11 and DR4 alleles were factors favouring longer survival.     

Altered DNA methylation in leukocytes with trisomy 21

The primary abnormality in Down syndrome (DS), trisomy 21, is well known; but how this chromosomal gain produces the complex DS phenotype, including immune system defects, is not well understood. We profiled DNA methylation in total peripheral blood leukocytes (PBL) and T-lymphocytes from adults with DS and normal controls and found gene-specific abnormalities of CpG methylation in DS, with many of the differentially methylated genes having known or predicted roles in lymphocyte development and function. Validation of the microarray data by bisulfite sequencing and methylation-sensitive Pyrosequencing (MS-Pyroseq) confirmed strong differences in methylation (p<0.0001) for each of 8 genes tested: TMEM131, TCF7, CD3Z/CD247, SH3BP2, EIF4E, PLD6, SUMO3, and CPT1B, in DS versus control PBL. In addition, we validated differential methylation of NOD2/CARD15 by bisulfite sequencing in DS versus control T-cells. The differentially methylated genes were found on various autosomes, with no enrichment on chromosome 21. Differences in methylation were generally stable in a given individual, remained significant after adjusting for age, and were not due to altered cell counts. Some but not all of the differentially methylated genes showed different mean mRNA expression in DS versus control PBL; and the altered expression of 5 of these genes, TMEM131, TCF7, CD3Z, NOD2, and NPDC1, was recapitulated by exposing normal lymphocytes to the demethylating drug 5-aza-2'deoxycytidine (5aza-dC) plus mitogens. We conclude that altered gene-specific DNA methylation is a recurrent and functionally relevant downstream response to trisomy 21 in human cells.     

Parental trisomy 21 mosaicism.

A family with three children with trisomy 21 in which the mother is a phenotypically normal, trisomy 21/normal mosaic was studied. Chromosome 21 fluorescent heteromorphisms were used to document that two of the three number 21's in two of the Down syndrome offspring were of maternal origin. Five cytogenetic surveys in which both parents of a child with trisomy 21 were studied have been reviewed. From these data, it is estimated that 3% of couples producing a child with trisomy 21 can be explained by parental mosaicism. From 17 informative sibships, with one parent mosaic, the segregation ratio was estimated to be 0.43 +/- 0.11.     

Differential behavior of cytotoxic effector cells against HLA antigens in strong genetic linkage disequilibrium

Five sets of cytotoxic effector cells were generated, using haploidentical, first degree relatives in five different families, against the HLA-A3; B7 serological determinants combined with different DR antigens. When tested against a panel of cells bearing combinations of the HLA-A, -B and -DR antigens it was shown that the HLA-B7 antigen was as strong a CML target determinant alone as it was in the presence of HLA-A3. The strength of the HLA-A3 antigen as target determinant varied. With effector cells primed to the HLA-A3; B7; DR2 haplotype, the A3 antigen alone behaved as a weak target determinant. When the same target cells were tested with the effector cells generated against HLA-A3; B7 without DR2, the A3 antigen behaved as a strong target determinant. A number of target cells lacking the serologically detectable HLA determinants present on the sensitizing HLA haplotype were identified as being killed by specific effector cells. These data suggest either a number of new CML target determinants controlled by different loci or the presence of a single, new locus with multiple alleles controlling CML targets.