Leonard H. Kellner

Methylenetetrahydrofolate reductase (MTHFR): The incidence of mutations C677T and A1298C in the Ashkenazi Jewish population

The polymorphic mutation C677T in the gene of MTHFR is considered a risk mutation for spina bifida and vascular disease. Another common mutation on the MTHFR gene, A1298C, has also been described as another risk mutation. We studied the frequencies of these two mutations on DNA samples from healthy Jewish individuals and compared them to the frequency of these mutations in DNA samples obtained from healthy individuals in South Texas. The presence of the C677T allele was determined by PCR and Hinf I digestion, and mutation A1298C by PCR and Mbo II digestion. A total of 310 alleles was examined for C677T in the Ashkenazi samples and 400 alleles in the non-Jewish samples. The rate of C677T among the Ashkenazi Jewish alleles was 47.7% as compared to 28.7% among the alleles from the non-Jewish population. The difference is statistically significant, P < 0.0005. Mutation A1298C was examined in 298 alleles of Jewish individuals and 374 alleles of non-Jewish counterparts from Texas. The rate of the A1298C mutation in the Jewish samples was 27.2% whereas in the non-Jewish was 35%. This was also statistically significant, P < 0.031. No individuals were homozygous for both mutations or were found to be homozygous for one mutation with heterozygosity of the other mutation, and that the C677T and the A1298C alleles did not occur in cis position. This study shows a unique distribution of C677T and the A1298C alleles among the Ashkenazi Jews. In spite of high frequency of C677T mutation, spina bifida is less common among Ashkenazi Jews. Further studies are needed to establish whether the C677T and the A1298C mutations have an impact on vascular disease in the Ashkenazi Jewish population.

First trimester screening for aneuploidy: serum biochemical markers.

The article reviews screening for Down syndrome in the first trimester (8-13 gestational weeks) with maternal serum analytes. In the first trimester, 2 serum markers stand out: pregnancy-associated plasma protein-A, a large glycoprotein tetramer, and free beta-human chorionic gonadotropin (beta-hCG), 1 of the 2 subunits of the glycoprotein hormone hCG. Some data indicate that hCG itself may be as effective as free beta-hCG in the first trimester. Maternal serum levels of pregnancy-associated plasma protein-A are low and free beta-hCG are high (consensus multiple of the medians, 0.4 and 1.8, respectively) in Down syndrome pregnancy. The consensus estimate of screening performance by using pregnancy-associated plasma protein-A and free beta-hCG in combination with maternal age is 60% detection rate at a 5% false positive rate. This is similar to the screening performance of second trimester double markers, but not as good as the screening performance of second trimester triple or quad markers. For this reason, first trimester screening with serum markers alone cannot be recommended except in cases in which second trimester screening cannot be done.

Quality assessment of routine nuchal translucency measurements: a North American laboratory perspective

Purpose: To assess nuchal translucency measurements that were performed as part of routine prenatal screening for Down syndrome.Methods: Collect ultrasound measurements of nuchal translucency and crown rump length provided by individual sonographers over a 6-month period to six North American prenatal screening laboratories, along with the laboratorys nuchal translucency interpretation in multiples of the median. For sonographers with 50 or more observations, compute three nuchal translucency quality measures (medians, standard deviations, and slopes), based on epidemiological monitoring.Results: Altogether, 23,462 nuchal translucency measurements were submitted by 850 sonographers. Among the 140 sonographers (16%) who submitted more than 50 observations, 76 (54%) were found to have all three quality measures in the target range. These 140 sonographers collectively accounted for 14,210 nuchal translucency measurements (61%). The most common single measure to be out of range was nuchal translucency multiples of the median, found for 29 of the 140 sonographers (21%).Conclusion: Laboratories should routinely monitor the quality of nuchal translucency measurements that are received for incorporation into Down syndrome screening risk calculations and interpretations. When possible, instituting sonographer-specific medians and providing individualized feedback about performance and numbers of women tested offer the potential to yield more consistent and improved performance.

Levels of urinary beta‐core fragment, total oestriol, and the ratio of the two in second‐trimester screening for Down syndrome

Levels of beta‐core fragment and total oestriol in second‐trimester maternal urine samples were measured in 32 Down syndrome pregnancies and 206 control pregnancies. Beta‐core fragment and total oestriol values were corrected for the urinary creatinine level and expressed as multiples of the control medians (MOM). In addition, the ratio of the beta‐core fragment level to the total oestriol level, without creatinine correction, was calculated, and expressed as MOM values. The median beta‐core fragment, total oestriol, and ratio levels in Down syndrome cases were 5·42, 0·64, and 9·32 MOM, respectively. In the Down syndrome pregnancies, 66 per cent of the beta‐core fragment levels were above the 95th centile of control levels, while 22 per cent of the total oestriol levels were below the fifth centile of control levels. In combination with maternal age, measurement of beta‐core fragment and total oestriol levels in Down syndrome pregnancy resulted in an 80 per cent detection rate at a 5 per cent false‐positive rate. Use of the ratio resulted in a univariate detection rate of 72 per cent. In combination with maternal age, the ratio resulted in a detection rate of 81 per cent at a 5 per cent false‐positive rate. Based on this unmatched study, the measurement of a ratio of beta‐core fragment to total oestriol levels, without the need for creatinine correction, may be useful in screening for fetal Down syndrome in second‐trimester urine.

Triple marker (α-fetoprotein, unconjugated estriol, human chorionic gonadotropin) versus α-fetoprotein plus free-β subunit in second-trimester maternal serum screening for fetal Down syndrome: A prospective comparison study

Abstract OBJECTIVE: Our purpose was to compare the efficacy of triple-marker screening (α-fetoprotein, unconjugated estriol, human chorionic gonadotropin) with α-fetoprotein plus free β-human chorionic gonadotropin. STUDY DESIGN: Free β-human chorionic gonadotropin was concurrently assayed in 2349 maternal serum samples. Trivariate and bivariate algorithms were used to calculate the risk for fetal Down syndrome by the two protocols. Free β-human chorionic gonadotropin from 12 cases of fetal Down syndrome previously screened with the triple marker was retrospectively assayed. RESULTS: Mean maternal age of our study was 29.8 years (range 14 to 51 years). The initial screen-positive rate with the triple marker was 8.0% compared with 12.8% for α-fetoprotein plus free β-human chorionic gonadotropin. All three cases of fetal Down syndrome ascertained in our prospective study were detected by the triple marker; in contrast, one of three was detected by α-fetoprotein plus free β-human chorionic gonadotropin. By adding 12 additional cases of fetal Down syndrome, 12 of 15 (80%) were screen positive with triple marker and nine of 15 (60%) were screen positive with α-fetoprotein plus free β-human chorionic gonadotropin. CONCLUSION: The detection rate of fetal Down syndrome was greater by use of a triple marker screen than when using α-fetoprotein plus free β-human chorionic gonadotropin. Our data do not support the claims of other studies that suggest that α-fetoprotein plus free β-human chorionic gonadotropin is superior to triple markers.

Comparison of 12 assays for detecting hCG and related molecules in urine samples from Down syndrome pregnancies

Urine is a new medium for Down syndrome testing. In an effort to determine the best type of human chorionic gonadotropin (hCG)‐related immunoassay for urine testing, we examined 14 Down syndrome and 91 unaffected pregnancy urine samples with 12 established assays. The assays included (a) those that detect hCG β‐core fragment only; (b) those that detect β‐core fragment with less than 18 per cent free β‐subunit cross‐reactivity; (c) that which equally detects free β‐subunit and β‐core fragment; and (d) those that detect hCG, free β‐subunit, or combinations thereof. The seven type a and b assays had the highest sensitivity for Down syndrome. The median MOM for Down syndrome was 5·93 (range 4·73–7·53). At a 10 per cent false‐positive rate, the median observed detection rate was 93 per cent (range 79–100 per cent) and the median predicted detection rate was 85 per cent (range 69–96 per cent). The assays that did not mainly detect β‐core fragment (types c and d) had poorer screening performance. The median MOM for Down syndrome was 2·70 (range 2·16–3·63 MOM). At a 10 per cent false‐positive rate, the median observed detection rate was 50 per cent (range 36–64 per cent) and the median predicted detection rate was 37 per cent (range 21–62 per cent). We infer that the assays that only detect β‐core fragment, or β‐core fragment with minor free β‐subunit cross‐reactivity (types a and b), are the better urine‐based tests for Down syndrome screening.

Prenatal screening for open neural tube defects

The era of prenatal screening for serious birth defects began in the 1970s with the discovery that amniotic fluid and maternal serum levels of alpha-fetoprotein (AFP) were increased in pregnancies affected by fetal open neural tube defects. Since then, prenatal screening has become a part of routine obstetric care. In this article, the use of AFP in prenatal screening for open neural tube defects is discussed in the context of the laboratory and the laboratorys interactions with the practicing obstetrician.

Noninvasive First-Trimester Screening for Fetal Aneuploidy

We reviewed all studies concerning noninvasive first trimester screening for fetal aneuploidy obtained from a MEDLINE search through June 1996 with additional sources identified through cross-referencing. Three screening and diagnostic modalities are of potential application in noninvasive first trimester testing for fetal aneuploidy: ultrasound, maternal biochemical markers, and analysis of fetal cells retrieved from maternal sources. Sensitivities of the sonographic finding of nuchal translucency thickness in combination with maternal age for trisomy 21, performed between 10 and 14 weeks of gestation in experienced hands, and maternal biochemical markers independently may be as high as 86 percent and 60 percent, respectively. Sensitivity, specificity, and predictive values of these diagnostic modalities alone, in combination with each other, or in conjunction with other predisposing factors such as maternal age, in large low risk populations have not currently been established. Analysis of fetal cells retrieved from maternal sources, although more complex, may offer definitive noninvasive prenatal diagnosis yet is not currently available in clinical practice. We conclude that noninvasive first trimester screening for fetal aneuploidy modalities including sonographic examination for nuchal translucency thickness and maternal biochemical markers, is feasible. Clinical feasibility; and all-encompassing clinical management paradigms of these and other early noninvasive first trimester screening methods for fetal aneuploidy, are not yet available.

Urinary analyte screening: a noninvasive detection method for Down syndrome?

Prenatal screening for Down syndrome using maternal serum markers achieves detection rates of 60-80% with a 5% false positive rate. Improvement in the accuracy of screening, as well as its ease and safety, will increase the use of such tests. The most effective of the current serum markers is human chorionic gonadotropin (hCG). Studies on beta core fragment (beta CF), the major urinary metabolite of hCG, have indicated that screening with beta CF and other markers measured in maternal urine might improve the detection of Down syndrome and provide a less expensive and simpler test. However, recent results have been unusually variable. Although it has great potential, the true clinical value of maternal urine screening to detect Down syndrome still remains to be determined.

Incorporation of fetal DNA detection assay in a noninvasive RhD diagnostic test

Noninvasive determination of fetal Rh genotype has become a standard part of clinical care. In general, noninvasive determination of Rh genotype relies on the amplification of fetal RhD gene sequences from DNA that is present in the mother’s plasma. In this context, the maternal DNA that is also present in the maternal plasma does not interfere with the test because the polymerase chain reaction (PCR) primers are carefully chosen so that they do not amplify from the Rh-negative mother. Thus, when RhD sequences are amplified, they are assumed to arise from fetal DNA and the fetus is deemed to be Rh positive. On the other hand, the prediction of an Rh-negative fetus is based on the failure to amplify RhD gene sequences, making this situation more problematic. Failure to amplify RhD may reflect the fact that the fetus is Rh negative, but it may result from technical issues such as lack of fetal DNA in the sample. To address this problem, most labs have relied upon the amplification of control targets, typically Ychromosome-specific sequences (e.g. SRY) that cannot be present in maternal DNA as a means of proving that fetal DNA is indeed present. Of course, Y-specific sequences are not present when the fetus is female, making it difficult to demonstrate the presence of fetal DNA in those pregnancies where the fetus is Rh negative and female. Therefore, a general method for assuring that fetal DNA is present in maternal plasma specimens would be very desirable. Some laboratories have incorporated tests for a panel of polymorphisms that involve insertion or deletion of DNA sequence, in an attempt to obtain a positive result derived from fetal, but not maternal, DNA (Zhou et al., 2005, Page-Christiaens et al., 2006). However, this method is expensive, timeconsuming and labor-intensive and is not particularly informative (Daniels et al., 2009). Another attempt at a universal test for presence of fetal DNA, using differential methylation present in the maspin (SERPINB5) promoter has been reported (Levine et al., 2004, Chim et al., 2005); however, the lack of sensitivity along with