Prenatal detection of free sialic acid storage disease: genetic and biochemical studies in nine families

Sialic acid storage disorders, Salla disease (SD) and a severe infantile form of disease (ISSD), are recessively inherited allelic lysosomal storage disorders due to impaired egress of free sialic acid from lysosomes. Fourteen pregnancies at risk of adult-type free sialic acid storage disease, SD, were monitored by sialic acid assays, genetic linkage or mutation detection analyses using chorionic villus samples. Three affected and 12 unaffected fetuses were identified. The first studies were based on the sialic acid assays alone, but the location of the gene enabled the use of genetic linkage analysis and, more recently, the identification of the SLC17A5 gene and disease-causing mutations added yet another possibility for prenatal studies. A missense mutation 115C→T (R39C) is present in 95% of all Finnish SD alleles, providing an easy and reliable means of diagnostic studies. Both molecular and biochemical (sialic acid assay) studies can be used for prenatal diagnosis of free sialic acid storage diseases. Copyright © 2001 John Wiley & Sons, Ltd.     

Identification of two mutations for ataxia telangiectasia among the Druze community

Ataxia telangiectasia (AT) is a rare autosomal recessive disease characterized by progressive cerebellar ataxia, immunodeficiency, susceptibility to lymphoreticular malignancies and cancer predisposition, hypersensitivity to ionic radiation and chromosomal instability. In this study, we report a founder effect of AT with two different mutations: 1339 C > T and 6672 del GG together with 6677 del TACG, found in four Israeli Druze clans originating from three different Druze centers in the Middle East (Lebanon, Syria and Jordan). The 1339 C > T mutation, which results in a stop codon at position 447 of the ATM protein, was observed in two unrelated clans originating from Lebanon and Jordan. The 6672 del GG/6677 del TACG mutation was observed in two unrelated clans originating from Syria and Lebanon. In the present study, simple and fast detection assays were developed for both mutations. The ability to identify AT carriers routinely provides a unique opportunity for prenatal diagnosis, genetic counseling as well as marriage guidance in the Druze community. Copyright © 2004 John Wiley & Sons, Ltd.     

Molecular genetic prenatal diagnosis for a case of autosomal recessive spondylocostal dysostosis

Autosomal recessive spondylocostal dysostosis type 1 (ARSCD1) is a member of the heterogeneous group of disorders termed the spondylocostal dysostoses that are characterized by multiple vertebral segmentation defects and rib anomalies. In these patients, the entire vertebral column is malformed and is replaced by multiple hemivertebrae giving rise to truncal shortening, abdominal protrusion and non-progressive spinal curvature. Genetic studies have shown that some cases of ARSCD are due to mutations in the somitogenesis gene, Delta-like 3 (DLL3), that encodes a ligand for the Notch signalling pathway—ARSCD type 1. To date, 17 different DLL3 gene mutations have been reported. A consanguineous family of Turkish origin with ARSCD type 1 due to a homozygous DLL3 mutation requested genetic prenatal diagnosis. Using DNA from a chorionic villus sample, both linkage analysis of the DLL3/19q region and direct sequencing for the familial mutation demonstrated that the unborn fetus was an unaffected carrier. This is the first case of molecular genetic prenatal diagnosis in any form of SCD. Copyright © 2003 John Wiley & Sons, Ltd.     

Prenatal molecular diagnosis in RASA1-related disease

RASA1-related disease is a rare autosomal dominant disease characterized by capillary malformations, arteriovenous malformations (AVMs), and/or arteriovenous fistulas (AFVs). Penetrance is nearly complete and vascular malformations may cause serious complications such as organ injury due to oxygenation disorder, brain abscess, hemorrhage, and stroke. Early diagnosis is useful in order to discuss optimal management, including AVMs/AVFs embolization or surgical procedures, and try to prevent some of the complications. In this context, molecular testing of RASA1 gene mutation in relatives may help to better manage the family. All arteriovenous malformations are however not accessible to such procedures. In addition, these therapeutic procedures may result in potential side effects and complications. A couple was referred to our genetics unit and asked us for prenatal genetic testing about a RASA1 mutation. Here, we discuss about arguments that led our team to accept prenatal testing. To the best of our knowledge, no molecular prenatal diagnosis was reported until now in RASA1-related diseases. This first report of prenatal diagnosis in RASA1-related diseases may also offer perspectives for a more general discussion in the field of inherited arteriovenous malformations.     

Prenatal exclusion of Leigh syndrome due to T8993C mutation in the mitochondrial DNA

Leigh syndrome (LS) is a mitochondrial encephalopathy that is caused by a mutation either in the mitochondrial DNA (mtDNA) or in the nuclear encoded genes of the mitochondrial proteins. Prenatal diagnosis of defects in the mtDNA is usually problematic because of mtDNA heteroplasmy and tissue specificity. However, the mutations T8993 G/C in the ATP synthase subunit 6 gene of the mtDNA show a more even tissue distribution and do not appear to change significantly over time. There are only few reports of prenatal diagnosis of the T8993G mutation in Leigh disease. Here we describe the first prenatal genetic testing of T8993C in a fetus of a mother whose previous child had died of Leigh syndrome due to the T8993C mutation. Mutant load in the chorionic villus sample (CVS) as well as in amniocytes was undetectable, thus predicting a very high likelihood of an unaffected outcome, indicative of a healthy baby. The diagnosis was confirmed after birth. Gathering data on the prenatal diagnosis of mtDNA mutations is of great importance so that prenatal diagnosis of both T8993G and T8993C mutations can be offered routinely. Copyright © 2002 John Wiley & Sons, Ltd.     

Non-invasive prenatal detection of achondroplasia in size-fractionated cell-free DNA by MALDI-TOF MS assay

Achondroplasia is the most common form of short-limbed dwarfism in humans and is caused by mutations in the FGFR3 gene. Currently, prenatal diagnosis of this disorder relies on invasive procedures. Recent studies have shown that fetal single gene point mutations could be detected in cell-free DNA (cf-DNA) from maternal plasma by either the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) assay with single allele base extension reaction (SABER) approach or the size fractionation of cf-DNA in maternal plasma. Here, we combined the two approaches to non-invasively examine the fetal G1138A mutation in maternal plasma. cf-DNA was extracted from maternal plasma samples obtained from two pregnant women at risk for achondroplasia. The fetal G1138A mutation was determined by the analysis of size-fractionated cf-DNA in maternal plasma using MALDI-TOF MS with SABER approach and homogenous MassEXTEND (hME) assay, respectively. The fetal G1138A mutation was detectable in the two achondroplasia-affected pregnancies by the analysis of cf-DNA in maternal plasma using MALDI-TOF MS. However, the size-fractionation approach led to a more precise detection of the fetal mutation in both analyses. This analysis would be suitable for non-invasive prenatal diagnosis of diseases caused by fetal single gene point mutations. Copyright © 2007 John Wiley & Sons, Ltd.     

Prenatal diagnosis of neurofibromatosis type 1: From flanking rflps to intragenic microsatellite markers

Even though the neurofibromatosis type 1 (NF1) gene was cloned more than 3 years ago, the process of identifying mutations has not been fruitful, and genetic counselling is mainly based on the use of linked markers. Since 1990, we have analysed 130 NF1 families and have performed six prenatal diagnoses. In each case, genetic counselling has relied on linked markers and informativity was achieved in all of them. The use of intragenic microsatellite polymorphisms (IVS27AAAT2.1, IVS27AC28.4, IVS27AC33.1, and IVS38GT53.0) has increased the informativeness in our series of NF1 families to an average of 90 per cent, providing accurate diagnosis and confirmation of the disease status.     

Prenatal diagnosis of β-thalassemia in Southern Punjab,Pakistan

Pakistan has a large population of more than 150 million people with an overall carrier frequency of approximately 5.6% for β-thalassemia. Punjab is the largest province of the country having more than 50% of the population. The state of β-thalassemia is alarming as consanguinity is very high (>81%) and the literacy rate is low in South Punjab. A thalassemia prevention program is the need of the hour in this part of Pakistan. In this study, we initiated awareness, screening, and characterization of the mutations causing β-thalassemia as well as a genetic counseling program mainly in the districts of Faisalabad and D.G. Khan to establish prenatal diagnosis, a facility previously unavailable in this region for disease prevention. A total of 248 unrelated transfusion-dependent children and the available members of their families were screened to characterize the mutations and identify the carriers. Genetic counseling was provided to these families and prenatal diagnosis offered. In the samples analyzed, 11 β-thalassemia mutations and three hemoglobin variants were detected mainly by using the Monoplex and Multiplex ARMS-PCR. First-trimester prenatal diagnosis was carried out through chorionic villus sampling (CVS) in seven pregnancies at risk. As a result of our campaign, 145 carrier couples planning to have more children gave their consent to have retrospective prenatal diagnosis in every pregnancy in future. A cooperative trend and a positive attitude toward the prevention of β-thalassemia were noticed in the families with affected children and in the general population. Copyright © 2006 John Wiley & Sons, Ltd.     

In vitro amplification of the α1-antitrypsin gene: Application to prenatal diagnosis

Discrimination of the M, Z, and S alleles of α₁-antritrypsin (AAT) has been carried out using in vitro gene amplification with the polymerase chain reaction (PCR). Amplification of 90 nucleotides surrounding the Z mutation site and 120 nucleotides surrounding the S mutation site dramatically improves the sensitivity and reliability of allele-specific oligonucleotide (ASO) hybridization for direct detection of these alleles. Analysis is performed using Southern blots or dot blots hybridized with 19 base oligonucleotides and differentially washed for allele specificity. Amplification of the Z and S mutation sites can be combined in one PCR to allow detection of both mutations when analysed by gel electrophoresis and Southern transfer. This technique can be performed reliably using less than 0·1 μg of genomic DNA or less than 100 amniocytes or white blood cells. This technique has been used to perform prenatal diagnosis on a chorionic villus sample (CVS) in a fetus at risk for the ZZ Pi type form of AAT deficiency.     

Prenatal ultrasound finding of atypical genitalia: Counseling,genetic testing and outcomes

Objective

To report uptake of genetic counseling (GC) and prenatal genetic testing after the finding of atypical genitalia on prenatal ultrasound (US) and the clinical and genetic findings of these pregnancies.

Methods

A retrospective cohort study (2017–2019) of atypical fetal genitalia in a large expert center for disorders/differences of sex development. We describe counseling aspects, invasive prenatal testing, genetic and clinical outcome of fetuses apparently without [group 1, n = 22 (38%)] or with [group 2, n = 36 (62%)] additional anomalies on US.

Results

In group 1, 86% of parents opted for GC versus 72% in group 2, and respectively 58% and 15% of these parents refrained from invasive testing. Atypical genitalia were postnatally confirmed in 91% (group 1) and 64% (group 2), indicating a high rate of false positive US diagnosis of ambiguous genitalia. Four genetic diagnoses were established in group 1 (18%) and 10 in group 2 (28%). The total genetic diagnostic yield was 24%. No terminations of pregnancy occurred in group 1.

Conclusions

For optimal care, referral for an expert fetal US scan, GC and invasive diagnostics including broad testing should be offered after prenatal detection of isolated atypical genitalia.     

Strategies for prenatal and preimplantation genetic diagnosis in Marfan syndrome (MFS)

Marfan syndrome (MFS) is an autosomal dominant disorder with a prevalence of 2–3 per 10 000 individuals. Symptoms range from skeletal overgrowth, cutaneous striae to ectopia lentis and aortic dilatation leading to dissection. Prenatal diagnosis was until recently mainly performed in familial cases by linkage analysis. However, mutation detection has become available with thorough screening methods. The phenotypic variability observed in MFS makes reproductive options difficult, as molecular diagnosis cannot predict clinical severity of the disease. Data are presented on 15 prenatal and/or preimplantation genetic diagnoses (PGD) in nine families, originating from Belgium, the Netherlands, Spain and France. In four families data from linkage analysis were used, whereas in five other families the causative FBN1 mutation was characterised. Four PGD cycles in two couples led to one ongoing pregnancy. In addition, two amniocenteses and nine chorionic villus (CV) samplings were performed. In five pregnancies an affected fetus was diagnosed. In one of them, the couple chose to continue the pregnancy and an affected child was born, whereas the other four couples decided to terminate the pregnancy. It is expected that the greater availability of mutation testing of the FBN1 gene will increase requests for prenatal diagnosis. PGD appears to be an acceptable alternative for couples facing ethical reproductive dilemmas. Copyright © 2002 John Wiley & Sons, Ltd.     

Prenatal diagnosis of Crigler–Najjar syndrome type I by single-strand conformation polymorphism (SSCP)

Crigler–Najjar syndrome type I (CN-I) is a rare and severe inherited disorder of bilirubin metabolism, caused by the total deficiency of bilirubin-UDP-glucuronosyltransferase (UGT) activity. Enzymatic diagnosis cannot be performed in chorionic villi or amniocytes as UGT is not active in these tissues. The cloning of the UGT1 gene and the identification of disease-causing mutations have led to the possibility of performing DNA-based diagnosis. Here we report DNA-based prenatal diagnosis of CN-I in two Tunisian families in whom CN-I patients were diagnosed. As we had previously shown that CN-I was, in Tunisia, associated with homozygosity for the Q357R mutation within the UGT1 gene, we were able to detect this mutation in both families and to show that it was easily recognized by single-strand conformation polymorphism (SSCP) analysis. In both cases, SSCP analysis of fetal DNA showed that the fetus was heterozygous for the Q357R mutation. In one family, the pregnancy was carried to term and a healthy baby was born, whereas, in the other family, the pregnancy is still continuing. Thus the prenatal diagnosis of CN-I is possible, provided disease-causing mutations have been identified. SSCP analysis of DNA prepared either from amniocytes or from chorionic villi is a simple, reliable and fast method for prenatal diagnosis. Copyright © 2002 John Wiley & Sons, Ltd.     

Prenatal diagnosis of free sialic acid storage disorders (SASD)

Free sialic acid storage disorders, Salla disease (SD) and Infantile sialic acid storage disease (ISSD), are lysosomal storage diseases due to impaired function of a sialic acid transporter, sialin, at the lysosomal membrane. Several mutations of the sialin gene, SLC17A5, are known, leading either to the severe neonatal/infantile disease or to the milder, adult-type developmental disorder, Salla disease. Free sialic acid accumulation in lysosomes causes increased tissue concentration and consequently elevated urinary excretion. Prenatal diagnosis of SASD is possible either by determination of free sialic acid concentration or by mutation analysis of the SLC17A5 gene in fetal specimen, in chorionic villus biopsy particularly. Both techniques have been successfully applied in several cases, sialic acid assay more often in ISSD cases but mutation analysis preferentially in SD. Sialic acid assay of amniotic fluid supernatant or cultured amniotic fluid cells may give erroneous results and should not be used for prenatal diagnosis of these disorders. The present comments are mainly based on our experience of prenatal diagnosis of SD in Finnish families. A founder mutation in SLC17A5 gene, 115C-> T, represents 95% of the disease alleles in the Finnish SD patients, which provides a unique possibility to apply mutation analysis. Therefore, molecular studies have successfully been used in 17 families since the identification of the gene and the characterization of the SD mutations. Earlier, eight prenatal studies were performed by measuring the free sialic acid concentration in chorionic villus samples. Copyright © 2006 John Wiley & Sons, Ltd.     

The amplification refractory mutation system (ARMS): A rapid and direct prenatal diagnostic technique for β-thalassaemia in Singapore

β-Thalassaemia major patients have chronic anaemia and since 3–4 per cent of Singaporeans carry the β-gene, prenatal diagnosis is essential. We evaluated the amplification refractory mutation system (ARMS) technique as a routine test for prenatal diagnosis of β-major. Six mutations along the β-gene were studied—41–42 (-TCTT), IVSII #654 (C-T), 17β (A-T), – 28 TATA (A-G), IVSI #5 (G-C), and IVSI #1 (G-T). Our results indicate that prenatal diagnosis using these mutations can be offered to 90 per cent (35/39) of our Chinese couples and 54·6 per cent (12/22) of our Malay couples at risk. Confirmation of ARMS results was carried out using allele-specific oligonucleotide hybridization. Prenatal diagnosis using ARMS was successfully carried out in nine cases which included a set of triplets and twins. The triplets were diagnosed with the β-trait carrying the 41–42 mutation. The couple with twins possessed the #654 mutation and one twin was diagnosed with the β-trait and the other with #654 homozygosity. Genomic sequencing of the undefined mutations in the Chinese couples revealed rarer mutations at − 29 and an ATG-AGG base substitution at the initiation codon for translation. In the Malay couples, genomic sequencing detected mutations at codon 15 (TGG-TAG) and codon 26 (GAG-AAG). We conclude that ARMS with its direct detection of amplified products by gel electrophoresis provides an accurate, rapid, and simpler method for our β-thalassaemia prenatal diagnosis programme in Singapore.     

The prenatal diagnosis of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) by mutation analysis

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an important cause of hereditary stroke. Mutations in the Notch3 gene are clearly causally linked to this progressive vascular disorder. Cerebral ischemic attacks, cognitive decline, strokes, and vascular dementia constitute the major manifestations of this disorder. This report details the prenatal detection of a Notch3 mutation in the fetus of a couple where the father had a known mutation in this gene. This is the first report of a prenatal diagnosis of CADASIL, and another example of a serious, highly penetrant, and relentlessly progressive degenerative genetic disorder presenting decades after birth and for which prenatal diagnosis is an option. Copyright © 2005 John Wiley & Sons, Ltd.     

Lesch—Nyhan syndrome: Carrier and prenatal diagnosis

We report the results of carrier and prenatal diagnosis for hypoxanthine guanine phosphoribosyltransferase (HPRT) deficiency, Lesch—Nyhan syndrome, by carrier testing of 83 women and prenatal analysis of 26 pregnancies. Our diagnostic methodologies include mutation detection and linkage analysis for probands and their families and biochemical measurement of HPRT enzyme activity for at-risk pregnancies. Identification of the mutation in the index case of each family permits precise carrier diagnosis using polymerase chain reaction (PCR) amplification of HPRT gene sequences and automated DNA sequencing. We demonstrate 100 per cent sensitivity for the detection of mutations in the HPRT gene of affected males and highly efficient carrier testing of at-risk females. Two other molecular methods proven to have high utility include PCR-based dosage analysis and linkage analysis by PCR amplification of a short tandem repeat (STR) in intron 3 of the HPRT gene. As a result, 45 at-risk women, 56 per cent of those tested, were identified not to be carriers of their family's HPRT gene mutation. Seven of these women were the mothers of affected males and prenatal testing for future pregnancies was recommended because of the possibility of gonadal mosaicism. Thirty-eight of these women were more distant relatives of affected males, thereby eliminating the need for future prenatal procedures. These studies illustrate the utility and precision of molecular methodologies for carrier and prenatal diagnosis of Lesch—Nyhan syndrome. These studies also illustrate that molecular diagnostic studies of affected males and carrier testing prior to pregnancy can clarify genetic risk predictions and eliminate unnecessary prenatal procedures.     

Prenatal diagnosis in a family with mitochondrial acetoacetyl-coenzyme a thiolase deficiency with the use of the polymerase chain reaction followed by the heteroduplex detection method

Mitochondrial acetoacetyl-coenzyme A (CoA) thiolase deficiency is an organic aciduria which affects isoleucine and ketone body catabolism. GK16 (the index patient) was affected with this disorder and previous studies had revealed that GK16 was a compound heterozygote with IVS8(+1) gt to tt and A301P mutations. In a subsequent pregnancy, prenatal diagnosis was performed and the fetus's amniocytes were analysed by the polymerase chain reaction (PCR) followed by the heteroduplex detection method on a Mutation Detection Enhancement gel. The fetus was identified as a carrier of the IVS8(+1) mutation. We confirmed the diagnosis by immunoblot analysis of extracted amniocytes and gene analysis with blood filter paper after delivery. This is the first report of prenatal diagnosis of this disorder at the gene level.     

New genetic and clinical evidence associated with fetal Beckwith–Wiedemann syndrome

Early detection of Beckwith–Wiedemann syndrome (BWS) is very important since it is very useful regarding counseling of parents concerning the risk of developing embryonic tumors, selection of the mode of delivery due to potential adrenal cysts that might bleed during labor, prevention of neonatal hypoglycemia and even options of pregnancy termination in non-viable fetuses. This report describes the prenatal classic sonographic triad of fetal BWS (omphalocele, macrosomia, macroglossia) and other supporting findings (hepatomegaly, adrenal enlargement) as well as additional postnatal evidence. Also, it demonstrates new molecular genetic evidence potentially associated with the disease (the presence of a novel heterozygous c.358G>T variant of the CDKN1C gene). Importantly, we provide new evidence indicating that elevated levels of the four serum biomarkers (alpha-fetoprotein, beta-human gonadotropin, unconjugated estriol, and inhibin-A) in late first or early second trimester might be strongly suggestive of BWS which may facilitate early detection especially in cases of no obvious anomaly. In conclusion, this study emphasizes on early detection of BWS as early as at 14 weeks of gestation, based on the abnormal rise of the four serum biomarkers together with omphalocele. To the best of our knowledge, this is the earliest prenatal detection of BWS ever reported. Finally, we provide new molecular genetic evidence that is, potentially associated with BWS.     

Rapid prenatal diagnosis of Duchenne muscular dystrophy with gene duplications by ion-pair reversed-phase high-performance liquid chromatography coupled with competitive multiplex polymerase chain reaction strategy

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are caused by various mutations in the dystrophin gene. Rapid prenatal diagnosis of DMD with gene duplications is difficult due to limitation in gene dosage determination and the requirement for a known disease-causing mutation in the pedigree to achieve a rapid and accurate diagnosis. We report, here, a case with rapid prenatal diagnosis of DMD-affected male with gene duplications in the absence of a known disease-causing variation in the pedigree by using ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) coupled with competitive multiplex polymerase chain reaction (PCR) protocol. In cases with clinical diagnosis of DMD/BMD, this test should identify greater than 92% of disease-causing DNA variants. The postnatal genetic diagnosis of this case and the same disease-causing mutations subsequently identified in other members of the pedigree confirmed the accuracy of competitive multiplex PCR/IP-RP-HPLC assay in direct prenatal diagnosis of DMD. Copyright © 2007 John Wiley & Sons, Ltd.