Analysis of β-globin gene haplotypes in Asian Indians: origin and spread of β-thalassaemia on the Indian subcontinent

-globin gene haplotypes were determined for 196 normal (-A) and 419 thalassaemia (-Th) chromosomes of individuals from four different regions of the Indian subcontinent; North-west Pakistan, Gujarat, Punjab and Sindh. Analysis of -A and -Th haplotypes and haplotype-mutation associations in each regional group along with a consideration of Indian history provided information about the origin and spread of -thalassaemia mutations on the Indian subcontinent. The data are consistent with relatively recent and local origins for most -thalassaemia mutations. The frequencies of particular alleles differ markedly in various regions and these may be useful population markers. Of the high frequency alleles, intervening sequence 1 (IVS-1) nucleotide 5 (G-C) and codons 41/42 (-CTTT) appear to be older as suggested by multiple haplotype associations and a widespread geographical distribution. The microepidemiology of -thalassaemia in this region reflects considerable ethnic diversity, gene flow from population migration and natural selection by malaria infection.     

Screening of mitochondrial mutations and insertion–deletion polymorphism in gestational diabetes mellitus in the Asian Indian population

In this study we scrutinized the association between the A8344G/A3243G mutations and a 9-bp deletion polymorphism with gestational diabetes mellitus (GDM) in an Asian Indian population. The A3243G mutation in the mitochondrial tRNA^Leu(UUR) causes mitochondrial encephalopathy myopathy, lactic acidosis, and stroke-like episodes (MELAS), while the A8344G mutation in tRNA^Lys causes myoclonus epilepsy with ragged red fibers (MERRF). We screened 140 pregnant women diagnosed with GDM and 140 non-GDM participants for these mutations by PCR-RFLP analysis. Both A3243G and A8344G were associated with GDM (A3243: OR-3.667, 95% CI = 1.001–13.43, p = 0.03; A8344G: OR-11.00, 95% CI = 0.6026–200.8, p = 0.04). Mitochondrial DNA mutations contribute to the development of GDM. Our results conclude that mitochondrial mutations are associated with the GDM women in our population. Thus it is important to screen other mitochondrial mutations in the GDM women.     

Identification of seven novel mutations in LH β-subunit gene by SSCP

Seven new point mutations have been identified from LH -subunit gene by PCR-mediated SSCP, and sequencing. One mutation was found changing amino acid from Gln₁₀₂ to Ser₁₀₂. The remaining six mutations, which did not change the codings, were in complete linkage disequilibrium. SSCP can be used in the diagnosis of LH-related disorders.     

Identification of 28 novel mutations in the Bardet–Biedl syndrome genes: the burden of private mutations in an extensively heterogeneous disease

Bardet–Biedl syndrome (BBS), an emblematic disease in the rapidly evolving field of ciliopathies, is characterized by pleiotropic clinical features and extensive genetic heterogeneity. To date, 14 BBS genes have been identified, 3 of which have been found mutated only in a single BBS family each (BBS11/TRIM32, BBS13/MKS1 and BBS14/MKS4/NPHP6). Previous reports of systematic mutation detection in large cohorts of BBS families (n > 90) have dealt only with a single gene, or at most small subsets of the known BBS genes. Here we report extensive analysis of a cohort of 174 BBS families for 12/14 genes, leading to the identification of 28 novel mutations. Two pathogenic mutations in a single gene have been found in 117 families, and a single heterozygous mutation in 17 families (of which 8 involve the BBS1 recurrent mutation, M390R). We confirm that BBS1 and BBS10 are the most frequently mutated genes, followed by BBS12. No mutations have been found in BBS11/TRIM32, the identification of which as a BBS gene only relies on a single missense mutation in a single consanguineous family. While a third variant allele has been observed in a few families, they are in most cases missenses of uncertain pathogenicity, contrasting with the type of mutations observed as two alleles in a single gene. We discuss the various strategies for diagnostic mutation detection, including homozygosity mapping and targeted arrays for the detection of previously reported mutations.     

Age and origin of the FCMD 3′-untranslated-region retrotransposal insertion mutation causing Fukuyama-type congenital muscular dystrophy in the Japanese population

Fukuyama-type congenital muscular dystrophy (FCMD), an autosomal recessive disorder with a high prevalence in the Japanese population, is characterised by severe muscular dystrophy associated with brain malformation (cortical dysgenesis) and mental retardation. In Japan, 87% of FCMD-bearing chromosomes carry a 3-kb retrotransposal insertion of tandemly repeated sequences within the disease gene recently identified on chromosome 9q31, and most of them share a common founder haplotype. FCMD is the first human disease known to be caused primarily by an ancient retrotransposal integration. By applying two methods for the study of linkage disequilibrium between flanking polymorphic markers and the disease locus, and of its decay over time, the age of the insertion mutation causing FCMD in Japanese patients is calculated to be approximately 102 generations (95% confidence interval: 86-117 g), or slightly less. The estimated age dates the most recent common ancestor of the mutation-bearing chromosomes back to the time (or a few centuries before) the Yayoi people started migrating to Japan from the Korean peninsula. This finding makes the molecular population genetics of FCMD understandable in the context of Japan's history and the founder effect consistent with the prevalent theory on the origins of the modern Japanese population.     

Novel promoter and splice junction defects add to the genetic,clinical or geographic heterogeneity of β-thalassaemia in the Portuguese population

Summary In order to delineate the spectrum and the relative abundance of -globin gene defects causing thalassaemia in the Portuguese population, a representative sample was analysed including 51 -thalassaemia carriers along with 26 patients representing different clinical phenotypes. Seven mutations were identified, four of which [codon 39 (CT), 39%; intervening sequence (IVS)1 nucleotide (nt) 1 (GA), 26%; IVS1 nt 110 (GA), 17%; IVS1 nt6 (TC), 15%] account for 97% of 93 -thalassaemia chromosomes. Two previously undescribed mutations, namely a CT substitution at position — 90 in the proximal CACCC box, and the deletion of nucleotides 4 and 5 (AG) in IVS 2 were identified. The uncommon, though ubiquitous, GT transversion at codon 121 was found once upon haplotype V. Direct prenatal diagnosis can be offered to 95% of couples at risk of bearing a thalassaemic child.     

Two novel missense mutations in the cystathionine β-synthase gene in homocystinuric patients

Direct sequencing of the coding region of the cystathionine -synthase (CBS) gene in two homocystinuric patients revealed the presence of two novel missense mutations. The first mutation, a 1111G A transition, resulted in the substitution of the evolutionary conserved valine-371 by a methionine residue (V371M) and created a new NlaIII restriction site. The second mutation, a GA transition at base-pair 494, resulted in an amino acid change from cysteine to tyrosine (C165Y) and abolished a BsoFI restriction site. Both mutations were found in a compound heterozygous state with the previously described 833T C transition.     

β-thalassemia mutations in the Portuguese population

Summary In this study we have carried out haplotype analysis on the -globin gene cluster and characterized the -thalassemia mutation by oligonucleotide hybridization in 14 patients with thalassemia major and 5 with sickle cell/-thalassemia originating from southern Portugal. We found that three mutations, namely the °-39, ° IVS-1 nt 1 and ⁺ IVS-1 nt 110 are prevalent accounting for 53%, 32% and 10% of the -thalassemia chromosomes respectively. In general each mutation was associated with a specific chromosomal haplotype; the ° mutation, however, was linked to three different haplotypes. These results indicate that three oligo-probes complementary to the most common mutations allow prenatal diagnosis by oligonucleotide analysis in 96% of the couples at risk of having offspring with thalassemia major in southern Portugal.     

Leftward deletion α-thalassaemia in the Saudi Arabian population

Summary Restriction endonucleases Bam HI and Bgl II were used to investigate the molecular basis of the deletion type of -thalassaemia in the Saudi population. Four homozygous cases and six heterozygous cases of the leftward deletion type of -thalassaemia (-) were identified. So far, the leftward type of -thalassaemia has been identified mainly in the Asiatic population, while the rightward deletion is universally distributed. This paper reports for the first time the presence of leftward deletion in the Saudi population and discusses the possibility of a more universal distribution of leftward deletion.     

Identification of novel mutations in the ABCA12 gene,c.1857delA and c.5653–5655delTAT,causing harlequin ichthyosis

Harlequin ichthyosis (HI) is a severe autosomal recessive developmental disorder of the skin that is frequently but not always fatal in the first few days of life. In HI, mutations in both ABCA12 gene alleles must have a severe impact on protein function and most mutations are truncating. The presence of at least one nontruncating mutation (predicting a residual protein function) usually causes a less severe congenital ichthyosis (lamellar ichthyosis or congenital ichthyosiform erythroderma). Here we report on a girl with severe HI diagnosed by prenatal ultrasound at 33 5/7 week gestation. Ultrasound findings included ectropion, eclabium, deformed nose, hands and feet, joint contractures, hyperechogenic amniotic fluid and polyhydramnion. After birth, palliative treatment was provided and she died on her first day of life. Sequence analysis of the ABCA12 gene identified two novel mutations, c.1857delA (predicting p.Lys619*) in exon 15 and c.5653–5655delTAT (predicting p.1885delTyr) in exon 37, each in heterozygous state. The c.5653–5655delTAT mutation is not truncating, but the deleted tyrosine at position 1885 is perfectly conserved among vertebrates and molecular studies evaluated the mutation as probably disease causing and damaging.     

Bovine β-defensins: Identification and characterization of novel bovine β-defensin genes and their expression in mammarygland tissue

-Defensin genes code for multifunctional peptides with a broad-range antimicrobial activity. In this project we hypothesized that -defensin genes may be candidate genes for resistance to mastitis. In this article we describe the identification and genomic characterization of eight bovine -defensin genes, including six novel defensin genes and two pseudogenes. Expression in the bovine mammary gland of one of the novel genes, DEFB401, has been demonstrated, as well as the expression of LAP, TAP, DEFB1, BNBD3, BNBD9, and BNBD12. For genomic characterization, 20 BACs from two different bovine BAC libraries (RZPD numbers 750 and 754) were isolated by PCR screening with -defensin consensus primers derived from published sequences. PCR products from BACs generated with consensus primers have been subcloned and sequenced, revealing a total of 16 genes and two pseudogenes. Six novel -defensin genes share the typical exon–intron structure and are highly homologous to published bovine -defensin genes. They are named DEFB401–DEFB405 and LAP-like, and two novel pseudogenes are named EBD-P and EBD-P2. Analysis of mammary gland tissue-derived cDNA from nine cows with different clinical findings demonstrated the expression of several -defensin genes mentioned above. First results indicate that the lactational status of the cow presumably has no influence on gene expression. Competent knowledge of antimicrobial activity of -defensins from literature, the abundance of -defensin mRNA in the bovine mammary gland, and the inducibility of some genes give first evidence that -defensins may play a role in local host defense during udder infections.The nucleotide sequence data reported in this article have been submitted to EMBL and have been assigned the accession numbers AJ563279–AJ563283, AJ567353–AJ567365, AJ567990–AJ567993, and AJ620296.     

The spectrum of mutations in erythrokeratodermias – novel and de novo mutations in GJB3

Intercellular channels in skin are a complex and functionally diverse system formed by at least eight connexins (Cx). Our recent molecular studies implicating Cx defects in inherited skin disorders emphasize the critical role of this signaling pathway in epidermal differentiation. Erythrokeratodermia variabilis (EKV) is an autosomal dominant genodermatosis with a striking phenotype characterized by the independent occurrence of transient localized erythema and hyperkeratosis. The disease maps to 1p34-p35, and recently we identified the causative gene GJB3 encoding Cx31. We have now investigated GJB3 in two families and three sporadic cases with EKV, and report three new heterozygous mutations. In a sporadic case, we detected a mutation leading to substitution of a conserved phenylalanine (F137L) in the third transmembrane domain, which likely interferes with the proper assembly or gating properties of connexons. In another family, all three affected individuals carried two distinct mutations on the same GJB3 allele. However, only a de novo heterozygous missense mutation replacing arginine 42 with proline (R42P) co-segregated with the disease, while a 12 bp deletion predicted to eliminate four amino acid residues in the variable carboxy terminal domain of Cx31 was also found in clinically unaffected relatives but not in 90 unaffected controls. Including the previously published mutations, in toto, five different missense mutations have now been detected in 6 out of 17 families investigated by our laboratory, all of which presumably affect the cytoplasmic amino terminal and transmembrane domains of Cx31. In contrast, two mutations linked to progressive high-tone hearing impairment were located in the second extracellular domain, suggesting that the character and position of Cx mutations determine their phenotypic expression in different tissues. However, the phenotypic spectrum of GJB3 mutations seems not to include progressive symmetric erythrokeratodermia, another dominant genodermatosis with overlapping features, since no mutations were found in six unrelated families tested.     

Molecular screening and fetal diagnosis of β-thalassemia in the Italian population

Summary This paper reports our experience of molecular screening and fetal diagnosis of -thalassemia in 457 at risk couples of Italian descent. Molecular screening was carried out by dot blot analysis on amplified DNA with oligonucleotide probes complementary to the eight most common mutations in Italians [39 (CT); 6 (-A); ⁺-87 (CG); ⁺ IVSI nt 110 (GA); IVSI nt 1 (GA); ⁺ IVSI nt 6 (TC); IVSII nt 1 (GA); ⁺ IVSII nt 745 (CG)]. By using this approach, we have been able to define the mutation in 92.8% of cases. The rest (all but four) were defined by direct sequencing and this led to the detection of nine rare mutations [76 (-C); ⁺ IVSI nt 5 (GA); ⁺ IVSI nt 5 (GC); ⁺ IVSI -1 (cod 30) (GC); ⁺-87 (CT), -290 bp del.; ⁺-101 (CT)], and to the characterization of a novel mutation consisting of the deletion of the G at the invariant AG of the IVSII splice acceptor site of the -globin gene ( IVSII nt 850-1 bp). In the remaining four cases, the -globin gene showed entirely normal sequences and the -globin gene cluster was intact, as indicated by Southern blot analysis. Fetal diagnosis was carried out by dot blot analysis with the oligonucleotide probes defined in the parents. The procedure is simple and reliable, and the results can be obtained within 1 week of sampling. No misdiagnosis has so far occurred. The results indicate that fetal diagnosis of -thalassemia by DNA analysis may be obtained in practically all cases (even in a population showing marked heterogeneity of -thalassemia) by the combination of dot blot analysis for detecting common mutations, and direct sequencing for defining those that are uncommon.     

Mediterranean types of β-thalassemia in the German population

Summary Forty -thalassemia genes from unrelated German heterozygotes with no known foreign ancestry were examined using the oligonucleotide technique and DNA restriction analysis, with the aim of determining the contribution of Mediterranean -thalassemia mutations to the prevalence of this trait in the German population. Of the 40 -thalassemia genes, 26 were identified as Mediterranean types (20 39 nonsense, 3 IVS2 nt 110, 2 IVS2 nt1, 1 IVS1 ntl GA). The geographic distribution of the birthplaces of the probands' grandparents revealed no difference in the proportion of Mediterranean and unidentified -thalassemia genes in the west and the north of Germany.     

Novel and recurrent mutations in the tyrosinase gene and the P gene in the German albino population

Albinism is a heterogeneous group of genetic disorders resulting from deficiencies in pigmentation. Clinically, it is divided into ocular (OA) and oculocutaneous albinism (OCA). OCA involves lack of pigment in the skin, hair, and eyes and results from mutations in the tyrosinase gene or in the P gene. OA mainly affects pigmentation in the visual system and may be a mild form of OCA or may be caused by other genetic defects. Clinical diagnosis of albinism type is difficult, because of the observed range of phenotypic variation. Thus, genetic analysis may be helpful with respect to a more accurate diagnosis. Here, we report the mutational profile, determined by genetic analysis of the tyrosinase and P genes, of a large German albino population. We have revealed a total of 42 distinct mutations, 19 of which are novel. Of the 74 unrelated patients screened, 32 (43%) had mutations in the tyrosinase gene, 16 (22%) had P gene mutations, and 26 (35%) patients had no detectable genetic abnormalities. This defines a population of albino patients who are tyrosinase-gene- and P-gene-negative and who thus may represent a good study group for searching for additional genes associated with albinism.     

Identification and expression of the medaka inhibin βE subunit

Activin E, a member of the TGF-β super family, is a protein dimer of mature inhibin βE subunits. Recently, it is reported that hepatic activin E may act as a hepatokine that alter whole body energy/glucose metabolism in human. However, orthologues of the activin E gene have yet to be identified in lower vertebrates, including fish. Here, we cloned the medaka (Oryzias latipes) activin E cDNA from liver. Among all the mammalian inhibin β subunits, the mature medaka activin E amino acid sequence shares the highest homology with mammalian activin E. Recombinant expression studies suggest that medaka activin E, the disulfide–bound mature form of mature inhibin βE subunits, may exert its effects in a way similar to that in mammals. Although activin E mRNA is predominantly expressed in liver in mammals, it is ubiquitously expressed in medaka tissues. Since expression in the liver was enhanced after a high fat diet, medaka activin E may be associated with energy/glucose metabolism, as shown in mice and human.

     

Cystathionine β-synthase 844Ins68 polymorphism is not associated with the levels of homocysteine and cysteine in an Indian population

Cystathionine β-synthase (CBS) is a key enzyme that plays a critical role in homocysteine metabolism and intracellular redox balance. We have analysed the association of the CBS 844Ins68 polymorphism alone and in combination with methylenetetrahydrofolate reductase (MTHFR C677T) and choline dehydrogenase (CHDH A119C) polymorphisms (the two polymorphisms recently shown to be associated with levels of homocysteine) with homocysteine, cysteine, folate and vitamin B₁₂ in 817 individuals (397 patients with coronary artery disease and 420 controls). The CBS 844Ins68 polymorphism alone or in combination with MTHFR C677T and CHDH A119C polymorphisms was not significantly associated with any of the biochemical variables studied.     

Identification of a novel splicing mutation and 1-bp deletion in the 17α-hydroxylase gene of Japanese patients with 17α-hydroxylase deficiency

We report studies of two unrelated Japanese patients with 17α-hydroxylase deficiency caused by mutations of the 17α-hydroxylase (CYP17) gene. We amplified all eight exons of the CYP17 gene, including the exon-intron boundaries, by the polymerase chain reaction and determined their nucleotide sequences. Patient 1 had novel, compound heterozygous mutations of the CYP17 gene. One mutant allele had a guanine to thymine transversion at position +5 in the splice donor site of intron 2. This splice-site mutation caused exon 2 skipping, as shown by in vitro minigene expression analysis of an allelic construct, resulting in a frameshift and introducing a premature stop codon (TAG) 60 bp downstream from the exon 1-3 boundary. The other allele had a missense mutation of His (CAC) to Leu (CTC) at codon 373 in exon 6. These two mutations abolished the 17α-hydroxylase and 17,20-lyase activities. Restriction fragment length polymorphism (RFLP) analysis with a mismatch oligonucleotide showed that the patient’s mother and brother carried the splice-site mutation, but not the missense mutation. Patient 2 was homozygous for a novel 1-bp deletion (cytosine) at codon 131 in exon 2. This 1-bp deletion produces a frameshift in translation and introduces a premature stop codon (TAG) proximal to the highly conserved heme iron-binding cysteine at codon 442 in microsomal cytochrome P450 steroid 17α-hydroxylase (P450c17). RFLP analysis showed that the mother was heterozygous for the mutation. Received: 15 November 1997 / Accepted: 15 March 1998