Conjoined twins in a monozygotic triplet pregnancy: prenatal diagnosis and X-inactivation

BACKGROUND: The etiology of monozygotic twinning is not known. Some investigators have implicated abnormal X-inactivation, which could also be related to the increased female:male ratio in higher order multiple gestations in general, and in monozygotic and conjoined twins (CTS) in particular. CTS are rare, and even more unusual when part of a triplet pregnancy. METHODS: DNA polymorphism analysis using 13 markers in the buccal cells of the triplets and the lymphocytes of the parents were used to evaluate zygosity. We investigated the X-inactivation pattern of the triplets by analyzing methylation at the androgen receptor gene. RESULTS: We found a female triplet gestation consisting of CTS and a normal singleton. The thoracopagus CTS were joined from the clavicles to the umbilicus. Congenital heart disease was suspected antenatally, but the precise delineation of the heart defects required extensive postnatal evaluation. There was a single placental mass with a thin dividing membrane. Cesarean section was carried out at 32 weeks after the onset of labor. Histologically, the placenta was diamniotic monochorionic. The normal singleton did well after delivery; the CTS died at 35 days from cardiopulmonary collapse. The babies were monozygotic (>99.99% probability). Each baby in this triplet set exhibited a random and symmetric X-inactivation pattern. The degree of X-inactivation skewing fell in the range of 50-65%. CONCLUSION: Genetic or environmental factors other than abnormal X-inactivation must be involved in causing monozygous multiple gestation or CTS. Despite prenatal diagnosis, shared myocardium or cardiac anomalies in CTS often determine the prognosis.     

Mutation carrier testing in Lesch-Nyhan syndrome families: HPRT mutant frequency and mutation analysis with peripheral blood T lymphocytes

Mutations in the X chromosome hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene are responsible for Lesch-Nyhan syndrome and related diseases in humans. Because the gene is on the X chromosome, males are affected and females in the families are at risk of being carriers of the mutation. Because there are so many different mutations that can cause the disease (218 different mutations in 271 families), genetic testing for carrier status of females requires detailed molecular analysis of the familial mutation. This analysis can be complicated by the unavailability of an affected male for study. In addition, when the mutation is a deletion (34 reported instances), molecular analysis in females is difficult because of the two X chromosomes. We have applied a peripheral blood T lymphocyte cloning assay that uses resistance to the purine analogue 6-thioguanine (TG) to measure the frequency of cells in females expressing a mutant HPRT allele to determine mutation carrier status in 123 females in 61 families. In families in which the HPRT mutation was determined and could be easily analyzed in samples from females, we found a mean (+/- SD) mutant frequency of 9.7 (+/- 8.7) x 10(-6) in noncarrier females and 2.9 (+/- 3.0) x 10(-2) in carrier females. The frequency in carrier females is less than the 0.5 expected for nonrandom X inactivation because of in vivo selection against HPRT mutation-expressing T lymphocytes or stem cells during prenatal development. The use of this cloning assay allows determination of the carrier status of females even when the HPRT mutation is not yet known or is difficult to determine in DNA samples from females. This approach provides a rapid assay that yields information on carrier status within 10 days of sample receipt.     

Genetic counselling, carrier detection, and prenatal diagnosis in hemophilia. A service experience

Our experience over three years (1984-1986) is described in carrier detection and prenatal testing for hemophilia. We have analysed 50 families: 37 hemophilia A and 13 hemophilia B, 22 isolated cases and 28 familial. Eighty-three women belonging to this panel asked for a genetic risk. Pedigree and coagulation studies were performed to estimate genetic risks according to the Bayesian method. At this point, 40% of the females at risk were recognized carriers before the DNA analysis. Molecular biology allowed the detection of only 7% more carriers and the exclusion of 34%. In 19% of the cases, it was impossible to estimate the genetic risk because the families were uninformative for the DNA polymorphisms used. Twenty-two prenatal diagnoses were performed; 3 affected male fetuses were recognized by DNA analysis and pregnancies were terminated. Eleven healthy boys were born.     

Skewed X-inactivation in a manifesting carrier of X-linked myotubular myopathy and in her non-manifesting carrier mother

X-linked recessive myotubular myopathy (XLMTM) is a muscle disorder usually affecting newborn males. In the majority of cases, muscle weakness and hypotonia lead to a rapid demise at neonatal age. The responsible MTM1 gene is located in proximal Xq28. Heterozygous carriers are described as being asymptomatic but, in a few cases, mild facial weakness has been reported. We report a family in which a 39-year old female showed severe progressive muscle weakness. XLMTM was initially diagnosed in the male offspring of one of the patient’s sisters. The patient, one of her sisters, and their mother were heterozygous carriers for a common MTM1 gene mutation. We found an extremely skewed X-inactivation pattern in the patient and, in the opposite direction, in her non-manifesting carrier mother, thus explaining her normal phenotype and indicating a possible inheritance of skewed X-inactivation. Linkage analysis excluded a possible involvement of the XIST locus at Xq13. Received: 2 November 1998 / Accepted: 7 January 1999     

First experiences in application of RFLP analysis for carrier detection in preparation of prenatal diagnosis of hemophilia A in the GDR

The application of the intragenic probe F8 e16-19 of factor VIII gene is reported for carrier detection in preparation of prenatal diagnosis of hemophilia A in a family at risk. The proband's mother is heterozygous for the Bcl I polymorphism and prenatal diagnosis can be offered to this family.     

Carrier detection and prenatal molecular diagnosis in a Duchenne muscular dystrophy family without any affected relative available.

In this paper we report a family where the affected DMD patients were not available for study and a molecular strategy was used for female carriers detection and for prenatal diagnosis. Linkage analysis was performed with two markers within the DMD gene, in all family members screened. DMD markers used (pERT87.8/Taq1 and pERT87.15/Xmn1) seemed not to be informative because the propositas mother (II-2) was homozygous for the minor allele at each marker (T2 and X2), however, the proposita and one sister carried only the major allele, which was inherited from the father. These results suggested that a deletion involving both markers could be present, and was inherited from the mother to both daughters. Quantitative multiplex PCR confirmed the deletion in female carriers, involving at least exons 12 to 17. DNA studies of cultured amniotic fluid cells at 14 weeks gestation, by amplification of specific Y-chromosome sequences, followed by multiplex PCR, lead to the diagnosis of a male fetus affected by DMD.     

Cosegregation of intragenic markers with a novel mutation that causes Crigler-Najjar syndrome type I: implication in carrier detection and prenatal diagnosis.

Crigler-Najjar syndrome type 1 (CN-1) is a familial disorder characterized by severe unconjugated hyperbilirubinemia and jaundice and leads to kernicterus, neurological damage, and eventual death unless treated with liver transplantation. Previous reports identified mutations in the UGT1 gene complex to be the cause of the disease. The total absence of all phenol/bilirubin UGT proteins and their activities in liver homogenate of a CN-1 patient was determined by enzymological and immunochemical analysis. A novel homozygous nonsense mutation (CGA-->TGA) was identified in the patient by the combined techniques of PCR and direct sequencing. This mutation was located in exon 3 of the constant region in the gene complex which is common to all phenol and bilirubin UGTs. The segregation of the mutation in the patient's family was analyzed and confirmed the recessive nature of the disease. Newly developed intragenic polymorphic probes (UGT1* 4 and UGT-Const) were used on Southern blots of MspI-digested genomic DNA of the patient and his family. The segregation of individual alleles within the family was observed from haplotypes generated. Comparison of the segregation of haplotypes with the mutation for the patient and his family revealed the allele identified by the A1-B1-C2 haplotype to be carrying the mutation. The risk of recombination occurring is negligible, because of the intragenic nature of the probes. This study demonstrates the potential usefulness of these probes in carrier detection and prenatal/presymptomatic diagnosis.     

Physical mapping identifies DXS265 as a useful genetic marker for carrier detection and prenatal diagnosis of X-linked agammaglobulinemia

The gene responsible for X-linked agammaglobulinemia (XLA) has not been identified; however, in the course of genetic linkage studies designed to map the locus more precisely, a number of closely linked polymorphic loci have been identified. These have proved to be useful in identifying carriers and in pre-natal diagnosis of this disease. The DXS178 locus was found to be closest to the XLA locus and has been the most usefully employed probe to date. Using physical mapping techniques, we have identified a previously cloned genetic marker, DXS265, as being situated within 5kb of DXS178. So far, we have found one family that is not informative for DXS178 but that is informative for DXS265; females in this family can now be offered the possibility of carrier determination and pre-natal diagnosis for this life-threatening disease.     

Hereditary tyrosinemia type I: strong association with haplotype 6 in French Canadians permits simple carrier detection and prenatal diagnosis.

Hereditary tyrosinemia type 1 (HT1), a severe inborn error of tyrosine catabolism, is caused by deficiency of the terminal enzyme, fumarylacetoacetate hydrolase (FAH). The highest reported frequency of HT1 is in the French Canadian population, especially in the Saguenay-Lac-St-Jean region. Using human FAH cDNA probes, we have identified 10 haplotypes with TaqI, KpnI, RsaI, BglII, and MspI RFLPs in 118 normal chromosomes from the French Canadian population. Interestingly, in 29 HT1 children, a prevalent haplotype, haplotype 6, was found to be strongly associated with the disease, at a frequency of 90% of alleles, as compared with approximately 18% in 35 control individuals. This increased to 96% in the 24 patients originating from Saguenay-Lac-St-Jean. These results suggest that one or only a few prevailing mutations are responsible for most of the HT1 cases in Saguenay-Lac-St-Jean. Since most patients were found to be homozygous for a specific haplotype in this population, FAH RFLPs have permitted simple carrier detection in nine different informative HT1 families, with a confidence level of 99.9%. Heterozygosity rate values obtained from 52 carriers indicated that approximately 88% of families at risk from Saguenay-Lac-St-Jean are fully or partially informative. Prenatal diagnosis was also achieved in an American family. Analysis of 24 HT1 patients from nine countries gave a frequency of approximately 52% for haplotype 6, suggesting a relatively high association, worldwide, of HT1 with this haplotype.     

一个中国遗传性血色病家系致病基因的突变分析

遗传性血色病(Hereditary hemochromatosis,HHC)是一种罕见的常染色体隐性遗传病。本课题组招募了一个HHC的近亲婚配家系,包括一名患HHC的先证者以及同一代的4名不患HHC的成员。通过对该HHC先证者进行全外显子组测序,在目前已知的与遗传性血色病相关的5个基因(HAMP、HJV、TFR2、FPN和HFE)中,发现在铁调素调节蛋白(Hemojuvelin,HJV)的编码基因HJV上存在两个纯合突变(c.G18C和c.GC962_963AA)。其中,前者能够引起HJV蛋白发生p.Q6H的改变,但该突变的危害性较小,可能与血色病的发病无关;后者能够引起HJV蛋白发生p.C321X的改变,从而翻译出缺失糖基磷脂肌醇锚定结构域的截短型HJV蛋白。除了HJV基因上的纯合突变外,该先证者还携带了其他12个纯合突变,但这些突变的危害性均不强且其所在基因的功能与铁代谢无关。本实验室内部测序数据显示,在一般中国人群中不存在p.C321X突变,提示HJV基因上的p.C321X纯合突变可能是该HHC患者的致病性突变。与此相一致的是,4名不患HHC的家系成员中该位点为野生型纯合子或杂合子,均非p.C321X纯合子。文章首次报道了HJV p.C321X纯合突变可导致HHC,该结果将有助于遗传性血色病的基因诊断和产前咨询。     

Prevention of homozygous beta-thalassemia by carrier screening and prenatal diagnosis in Sardinia.

We report here results of a 3-year pilot voluntary screening program coupled with prenatal diagnosis directed to the prospective prevention of homozygous beta-thalassemia (beta-thal) in Sardinia. The screening program took two approaches: outreach community testing and hospital testing on request after a period of sensibilization. The outreach testing was very effective as, taking into account the already known number of couples at risk with an affected proband (20), 74% of the couple at risk expected (61) on the basis of the carrier rate were identified. Less effective was the hospital testing in which half of the couples at risk expected were detected (502 with the 199 without an affected proband). After nondirective genetic counseling, approximately 85% of the couples at risk, which had a pregnancy, with no statistically significant difference between those with and those without a proband, requested prenatal testing. This figure showed a steadily increase from the beginning in 1977 to 1980. All the pregnancies (42), but two carrying homozygous fetuses, were terminated on parental request. A continuous hospital survey of thal-major admissions in the different hospitals of the counties showed a steady decline in the incidence figure at birth from 1976 (1:213) to 1978 (1:290). These results showed that even in a medium-developed, rural, Catholic population screening coupled with prenatal diagnosis can be successful in the control of a fatal, recessively inherited disorder.     

X-linked hypohidrotic ectodermal dysplasia: localization within the region Xq11-21.1 by linkage analysis and implications for carrier detection and prenatal diagnosis.

X-linked hypohidrotic ectodermal dysplasia (H.E.D.) is a disorder of abnormal morphogenesis of ectodermal structures and is of unknown pathogenesis. Neither relatively accurate carrier detection nor prenatal diagnosis has been available. Previous localization of the disorder by linkage analysis utilizing restriction-fragment polymorphisms, by our group and others, has placed the disorder in the general pericentromeric region. We have extended our previous study by analyzing 36 families by means of 10 DNA probes at nine marker loci and have localized the disorder to the region Xq11-Xq21.1, probably Xq12-Xq13. Three loci--DXS159 (theta = .01, z = 14.84), PGK1 (theta = .02, z = 13.44), and DXS72 (theta = .02, z = 11.38)--show very close linkage to the disorder, while five other pericentromeric loci (DXS146, DXS14, DXYS1, DXYS2, and DXS3) display significant but looser linkage. Analysis of the linkage data yields no significant evidence for nonallelic heterogeneity for the X-linked form of the disorder. Both multipoint analysis and examination of multiply informative meioses with known phase establish that the locus for H.E.D. is flanked on one side by the proximal long arm loci DXYS1, DXYS2, and DXS3 and on the other side by the short arm loci DXS146 and DXS14. Multipoint mapping could not resolve the order of H.E.D. and the three tightly linked loci. This order can be inferred from published data on physical mapping of marker loci in the pericentromeric region, which have utilized somatic cell hybrid lines established from a female with severe manifestations of H.E.D., and an X/9 translocation (breakpoint Xq13.1). If one assumes that the breakpoint of the translocation is within the locus for H.E.D. and that there has not been a rearrangement in the hybrid line, then DXS159 would be proximal to the disorder and PGK1 and DXS72 would be distal to the disorder. Both accurate carrier detection and prenatal diagnosis are now feasible in a majority of families at risk for the disorder.     

Mutation analysis of EXT1 and EXT2 Genes in a Korean family with multiple exostoses

A 35-year-old female patient diagnosed clinically as multiple exostosis visited the hospital for infertility evaluation and treatment. She had an operation in pelvis, humerus, tibia and femur in 1993. An extended pedigree analysis showed three of her siblings and several cousins have suffered from the same disease with a typical autosomal dominant pattern of inheritance. So she wanted a genetic test for her disease before having a child. For mutation analysis, DNAs were extracted from the patient and her brother. All exons and exon-intron boundaries of EXT1 and EXT2 genes were amplified by polymerase chain reactions. The PCR products were directly sequenced and analyzed by ABI genetic analyzer. A single base pair deletion c.2241delC in the exon 6 of EXT1 gene was detected in both patient and her brother. Generation of a premature stop codon resulting from frameshift of codons might be a causative of the disease. According to the human genome mutation data base (HGMD), the mutation detected is not previously documented.     

Mutation identification of the DSPP in a Chinese family with DGI-II and an up-to-date bioinformatic analysis

In this study, through linkage analysis of a four-generation Chinese family with multiple members afflicted with DGI (type II), we identified a novel missense mutation in DSPP. The mutation was located in exon 2 at the second nucleotide position of the last codon and resulted in a substitution of a proline with a leucine residue (c.50C>T, p.P17L, g.50C>T). To assess the potential effects of this novel mutation, we utilized various bioinformatics analysis programs. The results indicate that the mutation likely affects protein cleavage/trafficking. We also analyzed previously reported mutations of DSPP. In summary, our finding supports that the genomic sequence that corresponds to the P17 residue of DSPP is a mutational hotspot and P17 may be critical for the function of DSPP.     

Genomic carrier detection and prenatal diagnosis of haemophilia A in families at risk using the polymerase chain reaction (PCR).

The polymerase chain reaction (PCR) was applied in genomic analysis of families at risk for haemophilia A using the intragenic Bel I and Hind III polymorphism in introns 18 and 19, respectively, of factor VIII gene. For the latter the primers derived from exon 19 and 20 sequences allowed to amplify the whole intron 19 resulting in a 730 bp fragment. Hind III restriction of this fragment provides polymorphic fragments of 250 bp or 160 bp and 90 bp respectively. An also occurring 480 bp fragment can be used as internal control to circumvent misdiagnosis due to incomplete or failure of restriction. The Hind III polymorphism was successfully used in prenatal diagnosis of an affected male in the first trimenon of pregnancy. Fetal sexing was also performed by PCR technique using Y specific primers.     

一个中国B1型短指家系致病基因的突变分析

文章收集了一个中国B1型短指家系,通过连锁分析,发现该家系疾病的致病基因与ROR2基因连锁。PCR扩增ROR2基因突变热点区域后直接测序,在家系患者中发现一个c.2265C>A的杂合突变,该突变在蛋白质水平导致p.Y755X的改变,从而产生缺失部分结构域的截短ROR2蛋白,而在家系正常人以及家系外正常人中均未发现此突变。文章是国内首次报道B1型短指家系ROR2基因c.2265C>A突变,丰富了中国人ROR2基因突变谱。     

Population-based carrier screening and prenatal diagnosis of fragile X syndrome in East Asian populations

Identification of carriers of fragile X syndrome (FXS) with the subsequent prenatal diagnosis and knowledge of FXS-associated genetic profiles are essential for intervention in specific populations. We report the results of carrier screening of 39,458 East Asian adult women and prenatal diagnosis from 87 FXS carriers. The prevalence of FXS carriers and full mutation fetuses was estimated to be 1/581 and 1/3124 in East Asian populations, respectively. We confirmed the validity of the current threshold of CGG trinucleotide repeats for FMR1 categorization; the integral risks of full mutation expansion were approximately 6.0%, 43.8%, and 100% for premutation alleles with 55–74, 75–89, and ≥ 90 CGG repeats, respectively. The protective effect of AGG (adenine-guanine-guanine nucleotides) interruption in East Asian populations was validated, which is important in protecting premutation alleles with 75–89 CGG repeats from full mutation expansion. Finally, family history was shown not an effective indicator for FXS carrier screening in East Asian populations, and population-based screening was more cost-effective. This study provides an insight into the largest carrier screening and prenatal diagnosis for FXS in East Asian populations to date. The FXS-associated genetic profiles of East Asian populations are delineated, and population-based carrier screening is shown to be promising for FXS intervention.     

Mutation in a sex-determining gene in rainbow trout: detection and genetic analysis

In rainbow trout (Oncorhynchus mykiss), the acknowledged sex-determining system is genetic sex determination (GSD) with female homogamety (female symbolXX-male symbolXY). Subsequently, mitotic gynogens are all expected to be females. Unexpected maleness was fortuitously observed in a mitotic gynogenetic family of rainbow trout (13 males out of 27). An equal ratio of males and females suggested the possible segregation of some Mendelian sex-influencing factor. In order to perform a comprehensive analysis of the inheritance and expression of the factor involved, the transmission of maleness was studied across the next three generations, using both conventional and/or meiotic and mitotic gynogenetic offspring. On the whole, males as well as intersexes were observed in crosses between two expected carrier parents, and in gynogenetic offspring of expected carrier females, but not in crosses between one expected carrier parent and one normal XX control. Sex ratios in the different crosses often fitted Mendelian proportions, but not always. Both excess and lack of maleness were observed. The simplest hypothesis consistent with most results is a one-locus model, assuming the existence of a mutation (termed mal) of a sex-determining gene, which is able to override the primary XX mechanism of sex determination and to induce the development of testicular tissue in the gonads of expected XX individuals. The one-locus model requires that the mal mutation usually, but not systematically, behave as a recessive mutation and have a limited penetrance, that is, heterozygous (mal/+) may be sex reversed, homozygous (mal/mal) may remain female, and carrier individuals may undergo partial masculinization alone (many intersexes were recorded). Inconsistency in sex ratios among offspring of parents expected to respond the same way was recorded, indicating that other modifier loci may also be involved. Finally, the occurrence of both males and females in clonal progenies showed that epigenetic factors also likely influence the expression of maleness. The effects of the mal mutation are compared to similar mutations recently described in other fish species. The nature and location of the mal gene (carried by heterochromosomes or an autosomal pair) is briefly discussed in view of the knowledge recently acquired on the subject.     

An artifact band frequently associated with variable number of tandem repeat marker at phenylalanine hydroxylase gene: application in carrier detection and prenatal diagnosis of phenylketonuria

The variable number of tandem repeat (VNTR) marker located at the 3′-end of the phenylalanine hydroxylase (PAH) gene, PAHVNTR marker, is commonly used in carrier detection and prenatal diagnosis of the PKU disease. During the molecular diagnosis of the disease, an artifact band associated with the PAHVTNR marker was frequently observed. Analysis of genotyping data from nine trios families indicated that in heterozygote individuals, the observed stutter (artifact) bands at PAHVNTR marker were minor bands with one repeat sequence shorter than the upper main bands. More investigations using sequencing revealed that the artifact band was associated with VNTRs including seven or higher core repeats. In statistical analysis, 75% of the studied heterozygote individuals represented PCR artifact band. The presence of the artifact band associated with PAHVNTR marker highlights a serious alarm risk of possible technical misdiagnosis in the application of the PAHVNTR marker in carrier detection and prenatal diagnosis of the PKU disease.     

Linkage analysis of a large Latin-American family with X-linked retinitis pigmentosa and metallic sheen in the heterozygote carrier

An extended linkage analysis was performed on the large Latin-American kindred with X-linked retinitis pigmentosa (XLRP) and metallic sheen in the heterozygous carrier studied and reported previously by R.L. Nussbaum et al. (1985, Hum. Genet. 70:45-50) and on a smaller family with the same XLRP variant. In these kindreds the XLRP locus shows close linkage with Xp21 marker loci OTC and DXS206. The results of this linkage analysis agree with the observations made by Nussbaum et al. (1985) that an XLRP locus is distal to DXS7.