Linkage analysis of alpha 1-antitrypsin deficiency: lessons for complex diseases.

OBJECTIVES: Severe alpha 1-antitrypsin (A1AT) deficiency is the one proven genetic risk factor for chronic obstructive pulmonary disease (COPD). Familial aggregation has been demonstrated for COPD among individuals who do not have A1AT deficiency, but linkage analysis of COPD has not been reported. To investigate the optimal phenotype definitions and analytical methods for the linkage analysis of COPD, we examined a set of 28 A1AT- deficient families containing 155 individuals. We have used the protease inhibitor (PI) type as a genetic marker rather than a disease gene, and we have performed linkage analysis between PI type and serum A1AT level and spirometry-related phenotypes. METHODS: Linkage analysis was performed on the quantitative phenotypes forced expiratory volume at 1 s (FEV(1) as % predicted), the ratio of FEV(1) to forced vital capacity (FEV(1)/FVC as % predicted), and serum A1AT level using the variance component approach in SOLAR, the generalized estimating equation approach in RELPAL, and the model-based classical lod score method in LINKAGE. Linkage analysis with qualitative A1AT and spirometry phenotypes was performed using a model-based method (LINKAGE) and a model-free method (GENEHUNTER). Adjustments for smoking effects were investigated under each method. RESULTS: All of the methods demonstrated linkage of PI type to serum A1AT level. Interestingly, however, the other quantitative phenotypes provided only weak evidence for linkage of PI type to lung disease. Better evidence for linkage of lung disease to PI type was found using a moderate or a mild threshold for the definition of airflow obstruction. CONCLUSIONS: For linkage analysis of spirometry phenotypes in A1AT deficiency, qualitative phenotypes provided stronger evidence for linkage than quantitative phenotypes. Possible contributors to the stronger evidence for linkage to qualitative spirometry phenotypes include the ascertainment scheme and the nonnormality of the pulmonary function data in PI Z subjects. This study provides guidelines for studies of the genetics of COPD unrelated to A1AT deficiency.     

Low levels of beta hexosaminidase A in healthy individuals with apparent deficiency of this enzyme.

Appreciable beta hexosaminidase A (hex A) activity has been detected in cultured skin fibroblasts and melanoma tissue from healthy individuals previously reported as having deficiency of hex A activity indistinguishable from that of patients with Tay-Sachs disease (TSD). Identification and quantitation of hex A, amounting to 3.5%-6.9% of total beta hexosaminidase activity, has been obtained by cellulose acetate gel electrophoresis, DEAE-cellulose ion-exchange chromatography, radial immunodiffusion, and radioimmunoassay. Previous family studies suggested that these individuals may be compound heterozygotes for the common mutant TSD gene and a rare (allelic) mutant gene. Thus, the postulated rate mutant gene appears to code for the expression of low amounts of hex A. Heterozygotes for the rare mutant may be indistinguishable from heterozygotes for the common TSD mutant. However, direct visualization and quantitation of hex A by the methods described may prevent false-positive prenatal diagnosis of TSD in fetuses having the incomplete hex A deficiency of the type described in the four healthy individuals.     

Identification of a common mutation in patients with medium-chain acyl-CoA dehydrogenase deficiency

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is one of the most common recessively inherited metabolic diseases in man. We have studied fibroblast cultures obtained from three patients with MCAD deficiency by sequencing the entire coding region of MCAD mRNA. A single A to G nucleotide replacement which resulted in lysine329-to-glutamic acid329 substitution of the MCAD protein was identified in all cultures. Furthermore, this point mutation was present in 91% (31 of 34) of mutant MCAD alleles, indicating that the majority of cases with MCAD deficiency are caused by this type of mutation.     

Isolation of a new serotype of simian acquired immune deficiency syndrome type D retrovirus from Celebes black macaques (Macaca nigra) with immune deficiency and retroperitoneal fibromatosis.

A new serotype of simian acquired immune deficiency syndrome (SAIDS) retrovirus (type 2) belonging to the D genus of retroviruses is associated with a SAIDS occurring spontaneously in a colony of Celebes macaques (Macaca nigra) and rhesus macaques (Macaca mulatta) at the Oregon Regional Primate Research Center. This syndrome resembles SAIDS in M. mulatta at the California Primate Research Center, which is associated with a similar type D retrovirus (type 1). However, at the Oregon Center, SAIDS is distinguished by the occurrence of retroperitoneal fibromatosis in some of the affected monkeys. Type 2 virus was isolated from seven of seven macaques with SAIDS, retroperitoneal fibromatosis, or both and from one of six healthy macaques. The new strain is closely related to SAIDS retrovirus type 1 and Mason-Pfizer monkey virus but can be distinguished by competitive radioimmunoassay for minor core (p10) antigen and by genomic restriction endonuclease cleavage patterns. Neutralization tests indicate that type 1 and type 2 SAIDS retroviruses are distinct serotypes. Therefore, separate vaccines may be necessary to control these infections in colonies of captive macaques.     

A novel HPLC-based method to diagnose peroxisomal D-bifunctional protein enoyl-CoA hydratase deficiency

D-bifunctional protein (D-BP) plays an indispensable role in peroxisomal beta-oxidation, and its inherited deficiency in humans is associated with severe clinical abnormalities. Three different subtypes of D-BP deficiency can be distinguished: 1) a complete deficiency of D-BP (type I), 2) an isolated D-BP enoyl-CoA hydratase deficiency (type II), and 3) an isolated D-BP 3-hydroxyacyl-CoA dehydrogenase deficiency (type III). In this study, we developed a method to measure D-BP dehydrogenase activity independent of D-BP hydratase (D-BP HY) activity to distinguish between D-BP deficiency type I and type II, which until now was only possible by mutation analysis. For this assay, the hydratase domain of D-BP was expressed in the yeast Saccharomyces cerevisiae. After a coincubation of yeast homogenate expressing D-BP HY with fibroblast homogenate of patients using the enoyl-CoA ester of the bile acid intermediate trihydroxycholestanoic acid as substrate, D-BP dehydrogenase activity was measured. Fibroblasts of patients with a D-BP deficiency type II displayed D-BP dehydrogenase activity, whereas type I and type III patients did not. This newly developed assay to measure D-BP dehydrogenase activity in fibroblast homogenates provides a quick and reliable method to assign patients with deficient D-BP HY activity to the D-BP deficiency subgroups type I or type II.     

Mapping genetic loci for iron deficiency chlorosis in soybean

The objective of this study was to map genes controlling iron deficiency chlorosis in two intraspecific soybean [Glycine max (L.) Merrill] populations. Chlorosis symptoms were evaluated by visual scores and spectrometric chlorophyll determinations at the V4 stage (third trifoliolate leaf fully developed) in the field in 1993, and at V2 (first trifoliolate leaf fully developed) and V4 stages in 1994. A total of 89 RFLP and 10 SSR markers in the Pride B216 x A15 population, and 82 RFLP, 14 SSR and 1 morphological I (hilum color) markers in the Anoka x A7 population were used to map quantitative trait loci (QTL) affecting iron deficiency chlorosis. QTL with minor effects were detected on six linkage groups of the Pride B216 x A15 population, suggesting a typical polygene mechanism. In contrast, in the Anoka x A7 population, one QTL contributed an average of 72.7% of the visual score variation and 68.8% of the chlorophyll concentration variation and was mapped on linkage group N. Another QTL for visual score variation, and one for chlorophyll concentration variation were detected on linkage groups A1 and I, respectively. Due to the large LOD score and major genetic effect of the QTL on linkage group N, the quantitative data was reclassified into qualitative data fitting a one major gene model according to the means of the QTL genotypic classes. The major gene was mapped in the same interval of linkage group N using both visual scores and chlorophyll concentrations, thus verifying that one major gene is involved in segregation for iron chlorosis deficiency in the Anoka x A7 population. This study supported a previous hypothesis that two separate genetic mechanisms control iron deficiency in soybean.     

HPRTSardinia: a new point mutation causing HPRT deficiency without Lesch-Nyhan disease

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency always causing hyperuricemia presents various degrees of neurological manifestations, the most severe which is Lesch-Nyhan syndrome. The HPRT gene is situated in the region Xq26-q27.2 and consists of 9 exons. At least 300 different mutations at different sites in the HPRT coding region from exon 1 to exon 9 have been identified. A new mutation in the HPRT gene has been determined in one patient with complete deficiency of erythrocyte activity, with hyperuricemia and gout but without Lesch-Nyhan disease. Analysis of cultured fibroblasts revealed minimal residual HPRT activity mainly when guanine was the substrate. Genomic DNA sequencing demonstrated patient's mother heterozygosity for the mutation and no mutation in her brother. The mutation consists in a C-->T transversion at cDNA base 463 (C463T) in exon 6, resulting in proline to serine substitution at codon 155 (P155S). This mutation had not been reported previously and has been designated HPRT(Sardinia). The mutation identified in this patient allows some expression of functional enzyme in nucleated cells such as fibroblasts, indicating that such cell type may add further information to conventional blood analysis. A multicentre survey gathering patients with variant neurological forms could contribute to understand the pathophysiology of the neurobehavioral symptoms of HPRT deficiency.     

Identification of genetic mutations in Japanese patients with fructose-1,6-bisphosphatase deficiency.

Fructose-1,6-bisphosphatase (FBPase) deficiency is an autosomal recessive inherited disorder and may cause sudden unexpected infant death. We reported the first case of molecular diagnosis of FBPase deficiency, using cultured monocytes as a source for FBPase mRNA. In the present study, we confirmed the presence of the same genetic mutation in this patient by amplifying genomic DNA. Molecular analysis was also performed to diagnose another 12 Japanese patients with FBPase deficiency. Four mutations responsible for FBPase deficiency were identified in 10 patients from 8 unrelated families among a total of 13 patients from 11 unrelated families; no mutation was found in the remaining 3 patients from 3 unrelated families. The identified mutations included the mutation reported earlier, with an insertion of one G residue at base 961 in exon 7 (960/961insG) (10 alleles, including 2 alleles in the Japanese family from our previous report [46% of the 22 mutant alleles]), and three novel mutations--a G-->A transition at base 490 in exon 4 (G164S) (3 alleles [14%]), a C-->A transversion at base 530 in exon 4 (A177D) (1 allele [4%]), and a G-->T transversion at base 88 in exon 1 (E30X) (2 alleles [9%]). FBPase proteins with G164S or A177D mutations were enzymatically inactive when purified from E. coli. Another new mutation, a T-->C transition at base 974 in exon 7 (V325A), was found in the same allele with the G164S mutation in one family (one allele) but was not responsible for FBPase deficiency. Our results indicate that the insertion of one G residue at base 961 was associated with a preferential disease-causing alternation in 13 Japanese patients. Our results also indicate accurate carrier detection in eight families (73%) of 11 Japanese patients with FBPase deficiency, in whom mutations in both alleles were identified.     

Uncoupling phosphate deficiency from its major effects on growth and transcriptome via PHO1 expression in Arabidopsis

Inorganic phosphate (Pi) is one of the most limiting nutrients for plant growth in both natural and agricultural contexts. Pi‐deficiency leads to a strong decrease in shoot growth, and triggers extensive changes at the developmental, biochemical and gene expression levels that are presumably aimed at improving the acquisition of this nutrient and sustaining growth. The Arabidopsis thaliana PHO1 gene has previously been shown to participate in the transport of Pi from roots to shoots, and the null pho1 mutant has all the hallmarks associated with shoot Pi deficiency. We show here that A. thaliana plants with a reduced expression of PHO1 in roots have shoot growth similar to Pi‐sufficient plants, despite leaves being strongly Pi deficient. Furthermore, the gene expression profile normally triggered by Pi deficiency is suppressed in plants with low PHO1 expression. At comparable levels of shoot Pi supply, the wild type reduces shoot growth but maintains adequate shoot vacuolar Pi content, whereas the PHO1 underexpressor maintains maximal growth with strongly depleted Pi reserves. Expression of the Oryza sativa (rice) PHO1 ortholog in the pho1 null mutant also leads to plants that maintain normal growth and suppression of the Pi‐deficiency response, despite the low shoot Pi. These data show that it is possible to unlink low shoot Pi content with the responses normally associated with Pi deficiency through the modulation of PHO1 expression or activity. These data also show that reduced shoot growth is not a direct consequence of Pi deficiency, but is more likely to be a result of extensive gene expression reprogramming triggered by Pi deficiency.     

Selective IgA deficiency in autoimmune diseases

Selective immunoglobulin A deficiency (IgAD) is the most common primary immunodeficiency in Caucasians. It has previously been suggested to be associated with a variety of concomitant autoimmune diseases. In this review, we present data on the prevalence of IgAD in patients with Graves disease (GD), systemic lupus erythematosus (SLE), type 1 diabetes (T1D), celiac disease (CD), myasthenia gravis (MG) and rheumatoid arthritis (RA) on the basis of both our own recent large-scale screening results and literature data. Genetic factors are important for the development of both IgAD and various autoimmune disorders, including GD, SLE, T1D, CD, MG and RA, and a strong association with the major histocompatibility complex (MHC) region has been reported. In addition, non-MHC genes, such as interferon-induced helicase 1 (IFIH1) and c-type lectin domain family 16, member A (CLEC16A), are also associated with the development of IgAD and some of the above diseases. This indicates a possible common genetic background. In this review, we present suggestive evidence for a shared genetic predisposition between these disorders.     

Ochratoxin A-induced iron deficiency anemia.

Ochratoxin A at 8 micrograms per g of diet, but not at lower doses, fed to chickens from 1 day to 3 weeks of age resulted in significantly (P less than 0.05) decreased packed blood cell volume and hemoglobin concentration without altering the number of circulating erythrocytes. Serum iron and percentage of transferrin saturation were lowered at 4 and 8 micrograms/g. Therefore, anemia was characteristic of severe ochratoxicosis of young chickens, and the anemia was categorized as a hypochromic-microcytic anemia of the iron deficiency type. These data indicate that ochratoxin A by itself does not cause hemorrhagic anemia syndrome of chickens and that an anemia caused by a nutritional deficiency can be elicited by a mycotoxin.     

Ochratoxin A-induced iron deficiency anemia.

Ochratoxin A at 8 micrograms per g of diet, but not at lower doses, fed to chickens from 1 day to 3 weeks of age resulted in significantly (P less than 0.05) decreased packed blood cell volume and hemoglobin concentration without altering the number of circulating erythrocytes. Serum iron and percentage of transferrin saturation were lowered at 4 and 8 micrograms/g. Therefore, anemia was characteristic of severe ochratoxicosis of young chickens, and the anemia was categorized as a hypochromic-microcytic anemia of the iron deficiency type. These data indicate that ochratoxin A by itself does not cause hemorrhagic anemia syndrome of chickens and that an anemia caused by a nutritional deficiency can be elicited by a mycotoxin.     

Type VI collagen deficiency induces osteopenia with distortion of osteoblastic cell morphology

Bone consists of type I collagen as a major protein with minor various matrix proteins. Type VI collagen is one of bone matrix proteins but its function is not known. We therefore examined the effects of type VI collagen deficiency on bone. 3D-μCT analysis revealed that type VI collagen deficiency reduced cancellous bone mass. Cortical bone mass was not affected. Type VI collagen deficiency distorted the shape of osteoblasts both in the cancellous bone and in the cambium layer of periosteal region. Furthermore, type VI collagen deficiency disorganized collagen arrangement. These data indicate that type VI collagen contributes to maintain bone mass.     

Aldosterone synthase deficiency type I: hormonal and genetic analyses of two cases

Two female infants with clinical and biochemical findings of mineralocorticoid deficiency are presented. Both cases were diagnosed with aldosterone synthase type-I deficiency by multisteroid analysis. Two different mutations were located in the CYP11B2 gene: two homozygous amino acid substitutions (E188D and V386A) in case 1, and one heterozygous substitution (L324Q) and one heterozygous stop mutation (Y265X) in case 2.     

Fibroblast phosphodiesterase deficiency in Niemann-Pick disease.

Cultured skin fibroblasts from patients with Niemann-Pick disease types A and B were found to have diminished activity towards the synthetic substrates bis(4-methylumbelliferyl) pyrophosphate diester and bis(4-methylumbelliferyl) phosphate. Fibroblasts from a patient with Niemann-Pick disease type C exhibited less diminished activity. No reduction in activity was found towards bis(p-nitrophenyl) phosphate in types A, B or C fibroblasts. Maximum deficiency in types A and B fibroblasts was towards bis(4-methylumbelliferyl) pyrophosphate diester at pH 5.0, no deficiency being found at pH 7.2, either in the presence or absence of Mg⁺⁺ and cysteine.     

Radial transport of water and abscisic acid (ABA) in roots of Zea mays under conditions of nutrient deficiency

Radial water (J(V)) and abscisic acid (ABA) flows (J(ABA)) through maize root seedlings have been investigated under different conditions of nutrient deficiency. Whereas J(V) was reduced under nitrogen deficiency, potassium deficiency stimulated J(V). A substantial increase of J(ABA) was observed in roots kept under potassium deficiency. The observed changes of J(V) might have resulted from changed barrier properties of the endodermis. Nitrogen and potassium deficiency also caused an accumulation of endogenous ABA in root tissues. Under all conditions studied, except under K(+)-deficiency, external ABA (100 nM) caused an increase of J(V). The data of this study were used to analyse the relations between internal and endogenous root ABA, J(V), and J(ABA). The internal ABA of root tissues was positively correlated with J(V) and was highly significant (P <0.001 for internal and P=0.03 for endogenous root ABA) within the range 2-300 pmol g(-1) FW. It was also highly positively correlated to the radial ABA flows. There was also a highly positive correlation between J(V) and J(ABA). The data of this study indicate, for the first time, the relations between internal ABA, water, and ABA flows. Independent of treatment with external ABA, an ABA transport by solvent drag across the endodermis is confirmed.     

Leukocyte adhesion deficiency. Aberrant splicing of a conserved integrin sequence causes a moderate deficiency phenotype

Leukocyte adhesion deficiency (LAD) is a heritable deficiency of the LFA-1, Mac-1, p150,95 family of leukocyte alpha beta heterodimers (the leukocyte integrins). We have studied the defect in patients who synthesize an aberrantly small form of the beta subunit common to all three proteins. S1 nuclease protection showed the presence of a 90-nucleotide mismatch in RNA from patients and relatives, correlating with inheritance of the disease. Use of the Taq polymerase chain reaction to amplify this region of RNA after first strand cDNA synthesis and sequencing showed an in-frame deletion of 90 nucleotides in the extracellular domain. Thus, this highly conserved region, 63% and 53% identical in amino acid sequence to two other beta subunits of the integrin family, is required for association of the beta subunit with alpha subunits. The 90-nucleotide region corresponds to a single exon present in both the normal and patient genome. The patient DNA has a single G to C substitution in the 5' splice site. This results in the direct joining of nonconsecutive exons in an unusual type of abnormal RNA splicing. A small amount of normally spliced message, detected by S1 nuclease protection and Taq polymerase chain reaction, encodes a normal sized beta subunit which is surface-expressed and accounts for the low levels of leukocyte integrin expression observed in these patients, and hence the moderate phenotype.     

Epidemiology of alpha1-antitrypsin deficiency in the Netherlands

Summary During a 3-year period, newborns in the eastern part of the Netherlands were investigated for alpha₁-antitrypsin deficiency. Electroimmunoassay was used for screening, followed by Pi typing in suspected cases. In all 95 033 newborns were screened, and a mean frequency of deficiency (phenotypes PiZ, PiSZ, and PiS) of 8.00 in 10 000 was found.The distribution of deficient Pi types over the area was remarkably uneven, Pi type Z being more predominant north and Pi type S south of the Rhine. Cluster areas of alpha₁-antitrypsin deficiency, with frequencies of up to 59.6 in 10 000 liver births, occurred mainly in small rural communities. In urbanized areas the frequency of deficiency was lower than the mean.     

Genetic studies of B-lymphocyte deficiency and mastocytosis in strain A/WySnJ mice

The A/WySnJ mouse, but not the related A/J strain, has peripheral B-lymphocyte deficiency and mastocytosis. Minimally, two quantitative trait loci (QTLs) control the B-cell deficiency in (A/WySnJ x CAST/Ei)F2 intercross mice; one of them, Bcmd-1, mapped to Chromosome (Chr) 15. Several QTLs controlled the mastocytosis in this intercross, and it was not possible to determine whether any of them co-segregated with Bcmd-1. We have now mapped a second QTL controlling the B-cell deficiency, Bcmd-2, to Chr 4. Furthermore, we narrowed the map position of Bcmd-1 to <2.0 cM. Both QTLs have been confirmed through the construction of AW. Bcmd-1(c), AW. Bcmd-2(c), and AW. Bcmd-1(c)Bcmd-2(c) recombinant congenic strains. The Bcmd-1 locus is the major regulator of B-cell homeostasis, while Bcmd-2 is the minor regulator, and their effects are additive, as shown by splenic B-cells analysis in these congenic strains. In addition, Bcmd-2 or a linked locus controls mastocytosis, while Bcmd-1 does not, as indicated by splenic mast cell analysis in the congenic strains. Thus, the major genetic controls on B-cell homeostasis and mast cell homeostasis in A/WySnJ mice are asserted by distinct genes.