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Introduction: Chromosomal microarray analysis (CMA) is recognized as the first-tier test in the genetic evaluation of children with developmental delays, intellectual disabilities, congenital anomalies and autism spectrum disorders of unknown etiology. Array Design: To optimize detection of clinically relevant copy number variants associated with these conditions, we designed a whole-genome microarray, FirstStepDx PLUS (FSDX). A set of 88,435 custom probes was added to the Affymetrix CytoScanHD platform targeting genomic regions strongly associated with these conditions. This combination of 2,784,985 total probes results in the highest probe coverage and clinical yield for these disorders. Results and Discussion: Clinical testing of this patient population is validated on DNA from either non-invasive buccal swabs or traditional blood samples. In this report we provide data demonstrating the analytic and clinical validity of FSDX and provide an overview of results from the first 7,570 consecutive patients tested clinically. We further demonstrate that buccal sampling is an effective method of obtaining DNA samples, which may provide improved results compared to traditional blood sampling for patients with neurodevelopmental disorders who exhibit somatic mosaicism.

KBG syndrome is a rare autosomal dominant genetic condition characterized by neurological involvement and distinct facial, hand, and skeletal features. More than 70 cases have been reported; however, it is likely that KBG syndrome is underdiagnosed because of lack of comprehensive characterization of the heterogeneous phenotypic features. We describe the clinical manifestations in a male currently 13 years of age, who exhibited symptoms including epilepsy, severe developmental delay, distinct facial features, and hand anomalies, without a positive genetic diagnosis. Subsequent exome sequencing identified a novel de novo heterozygous single base pair duplication (c.6015dupA) in ANKRD11, which was validated by Sanger sequencing. This single-nucleotide duplication is predicted to lead to a premature stop codon and loss of function in ANKRD11, thereby implicating it as contributing to the proband's symptoms and yielding a molecular diagnosis of KBG syndrome. Before molecular diagnosis, this syndrome was not recognized in the proband, as several key features of the disorder were mild and were not recognized by clinicians, further supporting the concept of variable expressivity in many disorders. Although a diagnosis of cerebral folate deficiency has also been given, its significance for the proband's condition remains uncertain.

OBJECTIVE: To examine whether women with a personal or family history of preterm birth are more likely to have genetic variation in the human progesterone receptor (hPR). METHODS: Women with a singleton preterm birth at less than 37 weeks of gestation between 2002 and 2006 were identified from a prospectively collected clinical and biologic obstetric database (cases). Women in the control group were those with only term deliveries at or above 38 weeks of gestation. The Utah Population Database was queried for family history (first- or seconddegree relative) of preterm birth. DNA was extracted from stored buffy coats and genotyped for six single nucleotide polymorphisms in the hPR. RESULTS: One hundred fifty-four patients (92 women in the preterm case group, 62 women in the term control group) were included. All were white or Hispanic. There were no statistical differences between white and Hispanic allele frequencies. Women in the preterm case group were more likely to carry the minor allele, G (minor allele frequency 0.29 compared with 0.18, P.035) for rs471767, and were more likely to carry the GT haplotype across rs471767 and rs578029 compared with women in the term control group. Similar haplotype block variation was seen among women with preterm birth plus a family history of preterm birth. CONCLUSION: Allele and haplotype frequencies in the hPR are significantly different among women with preterm birth and women with preterm birth plus a family history of preterm birth. This suggests the hPR gene may be a candidate for association with preterm birth or familial preterm birth. (Obstet Gynecol 2010;115:765–70) LEVEL OF EVIDENCE: III

Objective: Obstetrical complications such as preeclampsia, fetal growth restriction, and placental abruption are associated with inadequate placental perfusion. Previous studies have shown that the angiotensinogen (AGT) Thr235 mutation is associated with abnormal remodeling of the uterine spiral arteries and occurs at higher frequencies in preeclampsia. This study was done to evaluate whether the AGT Thr235 mutation increases the risk of placental abruption. Materials and methods: We compared 62 placentas from women who had placental abruption with 240 control patients of similar age and ethnicity. DNA was extracted from paraffin blocks from placentas. AGT Met235Thr mutation status was determined by single fluoresceine labeled probe real-time PCR using a LightCycler system. Result: AGT genotypes were divided into three groups: MM (homozygous wild), TT (homozygous mutant), and MT (heterozygous). The constituent ratio of AGT genotype in abrupted placentas (MM 14.5%, MT 43.5%, TT 41.9%) was significantly different from in control group (MM42.5%, MT 39.6%, TT 17.9%) (p < 0.001). AGT mutant allele frequency in placental abruption (0.637) was significantly higher than in the control group (0.377) (p < 0.001). Conclusion: The AGT Thr235 mutation was observed more frequently in placental abruption. AGT Thr235 mutation may be considered a risk factor for placental abruption. Published by Elsevier Ltd.

High-resolution melting techniques are a simple and cost-effective alternative to other closed-tube genotyping methods. Here, we genotyped human platelet antigens (HPAs) 1 to 6 and 15 by high-resolution melting methods that did not require labeled probes. Conventional melting analysis with hybridization probes (HybProbes) was also performed at each locus. HybProbe assays were performed individually, whereas amplicon melting (HPAs 1 to 5 and 16) and unlabeled probe (HPA 6) assays were duplexed when possible. At all loci for each method, both homozygous and heterozygous genotypes were easily identified. We analyzed 100 blinded clinical samples (33 amniotic fluid, 12 cultured amniocytes, and 55 blood samples) for all 7 single-nucleotide polymorphisms (SNPs) by each method. Genotype assignments could be made in 99.0% of the SNPs by high-resolution melting and in 98.7% of the SNPs with HybProbes with an overall genotype concordance of 98.8%. Errors included two sample misidentifications and six incorrect assignments that were all resolved by repeating the analysis. Advantages of high-resolution melting include rapid assay development and execution, no need for modified oligonucleotides, and similar accuracy in genotyping compared with other closed-tube melting methods.

Problem: To determine if there is an association between two commonly inherited thrombophilias, the factor V Leiden and the G20210A prothrombin mutations, and fetal death. Method of study: We used a case–control study design to compare the frequencies of these mutations in women with fetal death and controls. Fetal death was the intrauterine death of the conceptus ≥10 weeks gestation. Controls had one live birth, no miscarriages, and no fetal death. Results were compared using chi square analysis. Results: One hundred and seventy-five cases and controls were identified. There were 4.6% of cases and 3.8% of controls heterozygous for the factor V Leiden mutation (NS), and 1.3% of cases and 1.7% of controls heterozygous for the prothrombin mutation (NS). Conclusion: In our population, neither the factor V Leiden nor the G20210A prothrombin mutations are associated with fetal death. Further evidence is required before routine screening for these mutations can be recommended.

HHT type 2 (HHT 2) is a multi-system vascular dysplasia caused by a mutation in the ALK-1 gene, but the phenotype has not been well defined. We report on 51 members of an HHT 2 kindred with an ALK-1 gene mutation shown to be associated with the disorder. This ALK-1 mutation was detected in 38 kindred members who were evaluated systematically for associated vascular abnormalities. Pulmonary arteriovenous malformations (AVMs) were found in 6% of those screened, cerebral AVM in 7%, hepatic AVM in 17%, and spinal AVM in 3%. We discuss these and other findings in the 38 affected kindred members, as well as findings in the 13 kindred members in whom the mutation was not detected. This study shows that pulmonary, cerebral, spinal, and hepatic AVMs can all occur in HHT 2. It also adds to the evidence suggesting that pulmonary AVMs are more common in HHT 1 than in HHT 2. We identify a higher prevalence of hepatic AVMs than previously reported in either HHT 1 or 2. This may be specific to the mutation in this kindred, but probably reflects the lack of routine screening for this manifestation. Even in this family in which all affected individuals have the same mutation, the clinical manifestations of HHT and their severity varied tremendously. Intrafamilial variation in expression of HHT is clearly significant, emphasizing the difficulty in establishing the diagnosis in individuals and in sub-typing families when DNA testing is not available.

STUDY DESIGN A cohort of 145 patients with adolescent idiopathic scoliosis (AIS) were identified and contacted to determine whether they had a family history of scoliosis. These results were submitted to an internal genealogical database to screen for potential connections to other AIS families. The severity and incidence of AIS in extended family groups were also analyzed. OBJECTIVES Our objectives were to quantify the genetic effect in AIS, determine the expressivity and penetrance of AIS in large family groupings, and examine larger scoliosis pedigrees for evidence of multiple genes. SUMMARY OF BACKGROUND DATA Previous reports have suggested an 80% connectedness among scoliosis families, but no clear evidence of multiple genes. It is not known if there are major gene(s). METHODS A cohort of 145 AIS probands were identified and contacted to ascertain whether they had a family history of AIS. Their medical records and spine radiographs were reviewed to confirm the diagnosis and determine the disease severity. Using an internal genealogical database, the cases were screened for potential connections that would produce larger extended pedigrees. RESULTS Overall, 131 of the probands were in the database and 127 showed connections to other scoliosis families, a 97% connectedness. These results suggest a major scoliosis gene, as more than 50% of the probands were connected by founders that all resided in England in the mid 1500s. The differences in penetrance (41% vs. 34%) and expressivity (38% vs. 61%) between seemingly unrelated large family groupings might suggest that two different genes are a major influence for AIS in these families. CONCLUSIONS Nearly all (97%) AIS patients have familial origins. There appears to be at least one major gene, and the differences in penetrance and expressivity in two large unconnected pedigrees might suggest the presence of more than one gene.

Objectives: The factor V Leiden mutation is the most common genetic predisposition to thrombosis. However, little is known concerning the reproductive outcome of mutation carriers or prenatal expressivity of this thrombogenic mutation. Our purpose was to examine whether this mutation presents phenotypically as miscarriage or idiopathic placental thrombosis. Study design: We performed two studies. First, a case-control comparison to determine whether fetal or maternal carriers of the factor V Leiden mutation are at risk for spontaneous miscarriage was performed, and, second, a cohort study evaluating placental infarction in fetuses carrying this mutation was performed. Results: We found a twofold increase in the factor V Leiden carrier frequency in 12 of 139 (8.6%) abortuses compared with 17 of 403 (4.2%) unselected pregnant women seen in the labor and delivery suite and, even more remarkable, a tenfold increase in the fetal carrier frequency in 10 of 24 (42%) placentas with >10% placental infarction compared with 7 of 372 (1.9%) placentas with Conclusions: These findings suggest a prenatal phenotype and effects of this mutation at the fetoplacental interface. If large prospective studies confirm these findings, then testing for this thrombogenic mutation should be considered in women and placental tissue from spontaneous abortuses and placentas with evidence of placental infarction. In addition to identifying individuals and families at risk for thrombosis, this information may help to improve our understanding of hemostasis and circulatory disturbances at the fetoplacental interface. (Am J Obstet Gynecol 1997;177:402-5.)

PROBLEM : The results regarding human leukocyte antigen (HLA) DQ α allele sharing in recurrent miscarriage couples are conflicting. The purpose of this study was to determine the frequency of HLA DQ α allele sharing in our unexplained recurrent spontaneous abortion (RSA) patients using modern DNA analytical techniques. METHODS : DNA was extracted from whole blood samples of 1) 51 couples with at least three miscarriages, and 2) 43 fertile couples (with at least seven children and no known history of recurrent miscarriage). The polymerase chain reaction (PCR) was used to amplify the second exon of the HLA DQ α locus on chromosome 6. Genotypes were identified by allele specific hybridization with 12 sequence-specific oligonucleotide probes. RESULTS : 47% of recurrent miscarriage couples and 35% of fertile couples shared no alleles. 47% of recurrent miscarriage couples compared to 58% of fertile couples shared one allele, and 6% of recurrent miscarriage couples and 7% of fertile couples shared two alleles. CONCLUSIONS : Reproductive partners with unexplained recurrent pregnancy loss have no increased frequency of HLA DQ α allele sharing. It is unlikely that HLA DQ α genotyping will be helpful in the management of patients with RSA.

The SCN8A gene encodes the sodium voltage-gated channel alpha subunit 8. Mutations in this gene have been associated with early infantile epileptic encephalopathy type 13. With the use of whole-exome sequencing, a de novo missense mutation in SCN8A was identified in a 4-yr-old female who initially exhibited symptoms of epilepsy at the age of 5 mo that progressed to a severe condition with very little movement, including being unable to sit or walk on her own.

Objective To determine whether polymorphisms in the FOXP3 gene are associated with preeclampsia. Study Design Case-control study in which 120 women with preeclampsia were compared with 120 healthy normotensive controls. Genetic variants (single nucleotide polymorphisms and microsatellites) in the FOXP3 gene were analyzed. Polymorphisms were chosen based on studies of the FOXP3 gene in other autoimmune disorders. Correction of P values for multiple comparisons was performed by using the Benjamini-Hochberg procedure. Results There were no differences in the genotypes or allele frequencies in the single nucleotide polymorphisms between cases and controls. The FOXP3 GT microsatellite allele at 266 bp was less common in cases than controls (1.0% vs 5.2%, P = .0264). However, this did not remain significant after correction for multiple comparisons. Conclusion Preeclampsia is not associated with FOXP3 gene polymorphisms that have been associated with other autoimmune disorders.

Study Design. Genetic association study investigating the association of genetic markers of melatonin signaling and biosynthesis with adolescent idiopathic scoliosis (AIS). Objective. To determine whether gene polymorphisms related to the melatonin signaling or biosynthesis pathways are associated with AIS. Summary of Background Data. Data have been published on the potential role of gene polymorphisms for melatonin receptor (MTNR) 1B in predicting AIS. Other genes in the melatonin pathways have been tested for association with AIS. Methods. The following genes involved in melatonin synthesis were evaluated herein: tryptophan 5-hyroxylase 1 (TPH1), serotonin N-acetyltransferase (SNAT), and hydroxyindoleo-methyltransferase (HIOMT). In addition, proteins involved in melatonin signaling were also included in this study: MTNR1A, MTNR1B, and protein kinase C delta (PKCδ). High throughput microarray-based single nucleotide polymorphism (SNP) genotyping was performed for these seven genes using DNA samples from 589 AIS subjects and 1533 ethnically matched controls. Chi-square analyses of allele frequency between AIS cases and controls were performed and odds ratios were calculated for all SNP markers. Results. Three SNPs were tested for both MTNR1A and HIOMT, 4 for TPH 1 and SNAT, 12 for PKCδ, and 7 for MTNR1B. The minor allele frequencies were not significantly different between AIS cases and controls. No association was thus found between AIS and the investigated SNPs. Conclusions. Genetic polymorphisms associated with either melatonin synthesis or its signaling pathway are unlikely to be commonly associated with AIS.

A heritability study of 69 extended Utah families with a history of adolescent idiopathic scoliosis (AIS) indicates that AIS is a polygenic, multifactorial condition. Each family reported a history of AIS within four generations; a total of 247 individuals were confirmed via X-rays and medical records to have AIS. Coefficient of kinship was more than 25 standard deviations higher for these 69 families than for the general population. Excluding all probands and assuming autosomal dominant inheritance, 1,260 individuals over the age of 16 were determined to be at risk for AIS because they have a parent with AIS. Assuming 50% of these individuals carry the allele, estimated penetrance in at-risk males is approximately 9%, and estimated penetrance in at-risk females is approximately 29%. Recurrence risk in relatives decreases as the degree of relationship to the affected individual becomes more distant; however, the lowest recurrence risk calculated, for third-degree relatives, is still an average of 9%, well above the general population's risk. Onset of AIS appears to be inherited separate from curve pattern and severity. In a study of phenotypes in 36 of the families, the affected individuals were consistent in either curve severity or curve pattern, but not both. It is unclear whether severity or pattern is more heritable, but it is possible that the location of the curve on the spine is the most heritable trait of the phenotype. The study demonstrates the genetic complexity of AIS, including the low penetrance of its cumulative alleles and variable expression.

Vascular endothelial growth factor-A (VEGFA) is normally expressed at high levels in the human placenta, and lower levels have been observed in placental tissue of women with recurrent pregnancy loss. The objective of this study was to determine if genetic polymorphisms in the VEGFA gene associated with altered gene expression play a role in some cases of recurrent pregnancy loss (RPL). A case–control study of 99 women with RPL and 181 fertile controls was performed evaluating four common VEGFA polymorphisms associated with altered gene expression (−2578 C/A, −1154 G/A, −634 G/C, and +936 C/T). The allele frequency of the −2578 A allele was lower among women with RPL compared to fertile controls (0.39 vs. 0.48, p =0.049), while the allele frequency of the −634 C allele was higher among women with RPL compared to fertile controls (0.39 vs. 0.29, p =0.020). Women with RPL and controls had similar allele frequencies for the −1154 and +936 minor alleles. We conclude that some allelic polymorphisms associated with altered expression of VEGFA are more common among women with RPL compared to fertile controls.

Objective Our objective was to determine whether polymorphisms in the promoter region of the interleukin-10 gene are more common in women with cervical insufficiency compared with controls. Study Design We conducted a case-control study. Histories and blood were obtained from 121 cases and 157 controls. DNA was extracted and purified by using Puregene kits. Samples were analyzed for the interleukin-10 –1082 G/A polymorphism and interleukin-10.G microsatellite in the interleukin-10 gene promoter region. Results The –1082 G/A polymorphism in the promoter region of the interleukin-10 gene occurred with similar frequency in cases and controls. The interleukin-10.G microsatellite contained 10 alleles (G6-G16). The G13 allele was present more frequently in cases (24.1%) compared with controls (14.6%) ( P = .05). Conclusion Interleukin-10 is known to down-regulate inflammation. The G13 allele in the interleukin-10.G microsatellite occurred more frequently in women with cervical insufficiency compared with controls, suggesting that alterations in inflammatory processes may play a role in cervical insufficiency.

Classic galactosemia is an autosomal recessive inherited error of galactose metabolism. It is caused by lack of galactose-1-phosphate uridyl transferase, an enzyme that is required to metabolize galactose-1-phosphate to uridine diphosphate galactose. The build up of galactose-1-phosphate is toxic at high levels and can damage the liver, brain, eyes, and other vital organs. Over 200 mutations have been identified in affected individuals. We describe an assay to identify nine target mutations or variants in the galactose-1-phosphate uridyl transferase gene, namely p.Q188R, p.S135L, p.K285N, p.L195P, p.T138M, p.Y209C, IVS2-2 A>G, p.L218L, and p.N314D. A single long-range PCR is followed by a multiplexed nucleotide extension assay (single nucleotide extension) and capillary electrophoresis to detect simultaneously all nine target mutations/variants. Fifty-four previously characterized samples (47 clinical samples and seven controls) gave a 100% concordance. We also report a nontarget novel mutation, p.L192X, and its profile using single nucleotide extension. This assay can complement the enzyme activity assay and identify familial mutations for testing additional family members.

Adolescent idiopathic scoliosis (AIS) is a clinically significant disorder with high heritability that affects 2-4% of the population. Genome-wide association studies have identified LBX1 as a strong susceptibility locus for AIS in Asian and Caucasian populations. Here we further dissect the genetic association with AIS in a Caucasian population. To identify genetic markers associated with AIS we employed a genome-wide association study (GWAS) design comparing 620 female Caucasian patients who developed idiopathic scoliosis during adolescence with 1,287 ethnically matched females who had normal spinal curves by skeletal maturity. The genomic region around LBX1 was imputed and haplotypes investigated for genetic signals under different inheritance models. The strongest signal was identified upstream of LBX1 (rs11190878, P(trend) = 4.18 × 10(-9), OR = 0.63[0.54-0.74]). None of the remaining SNPs pass the genome-wide significance threshold. We found rs11190870, downstream of LBX1 and previously associated with AIS in Asian populations, to be in modest linkage disequilibrium (LD) with rs11190878 (r(2) = 0.40, D' = 0.81). Haplotype analysis shows that rs11190870 and rs11190878 track a single risk factor that resides on the ancestral haplotype and is shared across ethnic groups. We identify six haplotypes at the LBX1 locus including two strongly associated haplotypes; a recessive risk haplotype (TTA, Control(freq) = 0.52, P = 1.25 × 10(-9), OR = 1.56), and a co-dominant protective haplotype (CCG, Control(freq) = 0.28, P = 2.75 × 10(-7), OR = 0.65). Together the association signals from LBX1 explain 1.4% of phenotypic variance. Our results identify two clinically relevant haplotypes in the LBX1-region with opposite effects on AIS risk. The study demonstrates the utility of haplotypes over un-phased SNPs for individualized risk assessment by more strongly delineating individuals at risk for AIS without compromising the effect size.

OBJECTIVE: To study the heritability of preterm delivery. METHODS: Women who delivered a singleton infant at less than 36 weeks of gestation were asked about their family history. Twenty-eight families were identified in which the proband had at least five first- or second-degree relatives with preterm delivery. An extensive genealogy database (GenDB) was constructed using more than 9,000 genealogy sources in the public domain (records before 1929). GenDB documents the relationships between more than 17.5 million ancestors and 3.5 million descendants of approximately 10,000 individuals who moved to Utah in the mid 1800s. This database was searched for the names, birth dates, and birthplaces of the four grandparents for each of the 28 probands. Pairwise coefficients of kinship were determined for the 93 preterm delivery grandparents identified, and for sets of 100 individuals born in the 1920s who were randomly selected from the population database. RESULTS: Probands had a mean of 3.3 grandparents included in this database. The average coefficient of kinship for controls was 1.5 x 10(6) (standard deviation = 0.6 x 10(6)). This measure agrees with previous calculations for the Utah population. The coefficient of kinship for familial preterm delivery grandparents was more than 50 standard deviations higher (3.4 x 10(5) [P < .001]). CONCLUSION: This study confirms the familial nature of preterm delivery. On average, gravidae randomly selected from our population are 23rd degree relatives, while these preterm delivery probands are eighth-degree relatives. A genome-wide scan using these affected families is underway.

To determine if there is an association between two commonly inherited thrombophilias, the factor V Leiden and the G20210A prothrombin mutations, and fetal death.We used a case-control study design to compare the frequencies of these mutations in women with fetal death and controls. Fetal death was the intrauterine death of the conceptusor =10 weeks gestation. Controls had one live birth, no miscarriages, and no fetal death. Results were compared using chi square analysis.One hundred and seventy-five cases and controls were identified. There were 4.6% of cases and 3.8% of controls heterozygous for the factor V Leiden mutation (NS), and 1.3% of cases and 1.7% of controls heterozygous for the prothrombin mutation (NS).In our population, neither the factor V Leiden nor the G20210A prothrombin mutations are associated with fetal death. Further evidence is required before routine screening for these mutations can be recommended.

Objective: Recent studies have identified hyperhomocysteinemia as a risk factor for both recurrent pregnancy loss and thrombosis. Antiphospholipid syndrome, an autoimmune disorder, is also characterized by recurrent pregnancy loss and thrombosis. Thus, our purpose was to determine if hyperhomocysteinemia is more common in patients with APS than normal fertile controls. Methods: Plasma, sera and whole blood were obtained from two groups of women: (1) 22 with well-characterized antiphospholipid syndrome; and (2) 41 healthy fertile controls. Levels of fasting homocysteine, vitamin B 6 , vitamin B 12 , folate and the incidence of the C677/T mutation of the methylene tetrahydrofolate reductase genotype (C677T/MTHFR) were determined. Results: The proportion of individuals with hyperhomocysteinemia and fasting plasma homocysteine levels were similar in women with APS and controls. Levels of vitamin B 6 , vitamin B 12 , folate and the incidence of C677/MTHFR were also similar in the two groups. Conclusion: Hyperhomocyteinemia and the C677T/MTHFR mutation are not common in women with antiphospholipid syndrome. Abnormal homocysteine metabolism is unlikely to play a major role in the pathogenesis of antiphospholipid syndrome.

Objectives The present paper reports the prenatal diagnosis of congenital adrenal hyperplasia (CAH) in two cases of 21-hydroxylase deficiency. DNA diagnostic errors can be caused by the presence of the highly homologous 21-hydroxylase pseudogene, CYP21P, adjacent to the functional gene, CYP21. The present paper details how complex gene conversions and rearrangements between the CYP21 and CYP21P pose unique complications for prenatal diagnosis. Methods Analysis of eight common mutations in the 21-hydroxylase gene as well as deletion of the entire gene is accomplished using polymerase chin reaction (PCR) followed by amplified created restriction site (ACRS) or allele-specific oligohybridization (ASO) and Southern blot followed by hybridization to a CYP21-specific probe. Linkage analysis was performed using microsatellite markers flanking the CYP21 gene. Results The direct mutation detection assay indicated a complicated gene conversion and rearrangement in the probands of both families. Interpretation of these rearrangements made it difficult to determine whether or not the fetuses would be affected with CAH. Linkage studies revealed that each fetus had inherited both parental disease chromosomes and was therefore predicted to be affected with CAH. Conclusion As observed in the two reported cases, direct DNA analysis may provide limited information due to gene conversion or rearrangement between the CYP21 and CYP21P genes. These cases suggest that direct mutation detection should be supported by linkage analysis, whenever possible, to provide more comprehensive information for the family. Copyright © 2002 John Wiley & Sons, Ltd.

Substantial evidence suggests that thrombosis contributes to the pathogenesis of primary pulmonary hypertension (PPH). An abnormal factor V (factor V Leiden) may contribute to thrombosis in the pulmonary microcirculation of PPH patients. A point mutation in which adenine is substituted for guanine at nucleotide 1691 (1691A) alters factor V so that it resists cleavage by activated protein C. Heterozygosity for the 1691A mutation is more common (2-8%) in Caucasian Europeans and Americans than in Africans (1%) and Asians (

Preeclampsia is associated with a common molecular variant of angiotensinogen (Met235Thr). This variant is in tight linkage disequilibrium with a mutation in the angiotensinogen promoter, G(-6)A, which leads to elevated expression in vitro. Since angiotensin II levels could play a role in atherotic changes of the uterine spiral arteries associated with preeclampsia, we investigated angiotensinogen expression in the first trimester uterus. We localized angiotensinogen transcription in uterine decidua using in situ reverse transcription PCR. We then compared decidual T235 expression levels to M235 levels in heterozygous women using an allele-specific ligation assay and a single nucleotide primer extension assay. In human decidua, angiotensinogen is expressed only in spiral artery smooth muscle cells. Heterozygous women have significantly elevated expression of the T235 allele compared to the M235 allele (P < 0.0001). These observations suggest that elevated expression of the T235 allele in decidual spiral arteries may cause first trimester atherotic changes leading to preeclampsia.

A common missense mutation in the factor V gene, the Leiden mutation, renders factor Va resistant to cleavage inactivation by activated protein C and predisposes patients to thrombotic events. We sought to evaluate the prevalence of the Leiden mutation and the associated thromboembolic events in a community hospital's low-risk obstetric population.Deoxyribonucleic acid was extracted from whole blood of 407 women. The polymerase chain reaction was used to amplify exon 10 of the factor V gene, followed by enzymatic digestion with MnI 1 for mutation detection. Medical charts were reviewed and patient characteristics, including age, gravidity, parity, obstetric complications, medical complications, and mode of delivery, were recorded.Fourteen of the 407 women carried the factor V Leiden mutation (13 heterozygotes and 1 homozygous mutant) for an allele frequency of 3%, consistent with the published carrier rate. Four of the 14 carriers (28%) had deep venous thrombosis, whereas the frequency of deep venous thrombosis in this obstetric population was1%. Another patient carrying the mutation had a consumptive coagulopathy of unknown etiology at 20 weeks' gestation, necessitating delivery.The Leiden mutation is relatively common in the general obstetric population. The high rate of deep venous thrombosis noted in our series suggests the need for genetic testing for this mutation in women with a thrombotic event during pregnancy.

OBJECTIVE: A recent study showed that resistance to activated protein C may underlie some cases of severe preeclampsia. A common missense mutation in the factor V gene, the Leiden mutation, is the most frequent genetic cause of resistance to activated protein C. Our objective was to determine whether this mutation is more prevalent in patients with severe preeclampsia than in normotensive controls. STUDY DESIGN: Deoxyribonucleic acid was extracted from whole blood of 158 gravid women meeting criteria of The American College of Obstetricians and Gynecologists for severe preeclampsia and 403 normotensive gravid women. The polymerase chain reaction was used to amplify exon 10 of the factor V gene, followed by allele-specific restriction with Mnl 1 for mutation detection. Results were analyzed with a χ 2 contingency table. RESULTS: No patients were homozygous for the Leiden mutation. Fourteen of 158 women with severe preeclampsia (8.9%) were heterozygous for the Leiden mutation compared with 17 of 403 normotensive gravid controls (4.2%). The difference in frequency between women with severe preeclampsia and normotensive controls was statistically significant, χ 2 4.686, p = 0.03. CONCLUSIONS: Our data suggest that carriers of the factor V Leiden mutation are at increased risk for severe preeclampsia. Deoxyribonucleic acid analysis for the factor V Leiden mutation could serve as one component of a genetic screening profile for preeclampsia and other adverse pregnancy outcomes. Women who carry this mutation are at increased risk for deep venous thrombosis. Carriers of this common thrombophilic mutation may be identified so that adequate counseling regarding future contraceptive usage and effective thromboembolic prophylaxis during pregnancy and surgical procedures may be offered. (Am J Obstet Gynecol 1996;175:902-5.)