American Journal of Medical Genetics 16519425 (1983)

Phenotypic Heterogeneity in Neural Tube Defects: A Clue to Causal Heterogeneity

R.A. Martin, R.M. Fineman, and L.B. Jorde

Division of Medical Genetics, Department of Pediatrics, University of Utah Medical Center, Salt Lake City

We report here retrospective data on 991 liveborn and stillborn infants with neural tube defects (NTDs) born to Utah residents from January 1, 1940 to December 3 1 , 1979. Data were obtained from multiple sources including approximately 1.25 million vital statistics records and several hundred physician and hospital charts. Causal heterogeneity among NTD patients is presumed because 6% of our cases have other congenital anomalies not part of the NTD field defect. A significant association of NTDs with oral clefts is noted. Sex ratios and empiric recurrence risks for isolated NTDs and NTDs associated with other major malformations are also calculated.

Key words: neural tube defects, spina bifida, anencephaly, heterogeneity, midline, schisis associ- ation, multiple malformations, multiple ascertainment

INTRODUCTION

Many investigators have examined possible genetic and environmental causes of NTDs. Elwood and Elwood [1980] reviewed a large number of these investiga- tions. Of note in these studies is that NTDs are usually separated into just two major groups-anencephaly and spina bifida, although some researchers place encephalo- celes into a third category. Only in recent studies have patients with isolated NTDs been separated from NTD patients having additional malformations beyond the primary NTD [Holmes et al, 1976; Chance et al, 1981; Khoury et al, 1982a,b]. This separation is appropriate since many known conditions that include an NTD have epidemiologic and genetic patterns that differ from those of isolated NTDs (eg, Meckel syndrome, amnion-rupture sequence, oculoauriculo-vertebral dysplasia, tri- somy 18, trisomy 13). We document here the phenotypic heterogeneity in a large, multiply ascertained NTD population and present data that support causal heteroge- neity of this group.

Received for publication Janurary 30, 1983; revision received June 13, 1983.

Address reprint requests to Rick A. Martin, Department of Pediatrics, Division of Medical Genetics, University of Utah Medical Center, Salt Lake City, Utah 84132.

0 1983 Alan R. Liss, Inc.

520 Martin, Fineman, and Jorde

METHODS

With permission of the Utah Bureau of Vital Statistics we reviewed all Utah birth, death and stillbirth (fetuses past 20 week gestation) certificates from January 1 , 1940 to December 31, 1979. During this period 979,873 liveborn infants, 248,208 deaths, and 11,161 fetal deaths were recorded. Additionally, charts from the 9 major Wasatch front (Ogden, Salt Lake City, and Provo) hospitals, office records of 15 were reviewed for the same 40-year period (totalling at least 300 NTD cases). Epidemiologic and available birth defect information was collected in each case. These sources were reviewed in the following order: (1) birth certificates, (2) stillbirth certificates, (3) physician’s office, hospital and HCS records, and (4) death certificates. Available information was listed on our standard birth defects registry card and entered into our computerized NTD registry. The registry was then linked to the Utah Genealogical Data Base [Skolnick, 19801 for calculation of recurrence risks.

NTD cases were divided into five groups: (1) spina bifida (includes myelomen- ingocele, meningocele, and rachischisis; excludes spina bifida occulta) , (2) anence- phaly (includes cranioschisis), (3) encephalocele (includes midline exencephaly, cranium bifidum, and encephalomeningocele), (4) craniorachischisis, (5) double NTDs (spina bifida with anencephaly, spina bifida with encephalocele or two spina bifida lesions). In each group, cases with apparently isolated NTDs were separated from those with other major malformations. Major malformations, as defined here, exclude those anomalies considered to be secondary to the primary NTD lesion such as club feet, dislocated hips, lower limb flexion contractures, and hydrocephalus. Minor anomalies such as hydroceles and clinodactyly were not considered. In addition to classifying and describing cases, we analyzed prevalence and sex ratio data, cleft lip and/or cleft palate association with NTDs, the association of double NTDs with other major malformations versus single NTDs with other major malformations, and the significance of the difference in percentage of NTDs with malformations in our study versus that of Khoury et a1 [1982a].

RESULTS Data Sources

Table I shows the number and types of NTDs ascertained from each data source. The sources are listed vertically in the order reviewed. Cases ascertained from more than one source are included in the figure where they were first ascertained. Review of birth certificates yielded 518 cases or 52.3% of the total sample while stillbirth certificates accounted for an additional 296 cases (29.9%), most of these being anencephalics as expected. Hospital/physician/HCS records and death certificates are responsible for the remaining 177 cases (9.5% and 8.4%, respectively).

Classification

Classification of NTDs into the five previously mentioned groups is shown in Table 11. There were 930 cases (94%) of isolated NTDs. In six cases there were “multiple congenital anomalies” but the anomalies were never identified; thus , they were placed in the isolated category. Seven cases, first identified as isolated NTDs from Vital Statistics records, were changed to the multiple malformation category

Neural Tube Defect Heterogeneity 521

TABLE I. Sources of Data

Source Anencephaly Spina Bifida Encephalocele Total

Birth certificates 117 359 42 518 (52.3%) 239 50 7 296 (29.9%) Stillbirth certificates

17 60 6 83 (8.4%) Death certificates Total 374 547 70 99 1

Note: (1) Double NTDs counted as either anencephaly, spina bifida, or encephalocele depending on which was listed first in record; (2) craniorachischisis counted as anencephaly.

Hospital/physician/HCS records 1 78 15 94 (9.5%)

TABLE 11. NTD Classification

With Isolated CL With MCA (with or Isolated and/or CPb without CL and/or CP)b Total

Type of NTD number (%)" number (%)" number (%)" number (%)"

Spina bifidairachischisis 506 (51) 9 (1) 27 (3) 542 (55) Anencephaly/cranioschisis 329 (33) 6 (1) 4 (1) 339 (34) Encephalocele 58 (6) 0 8 (1) 66 (7) Craniorachischisis 8 (1) 0 1 (1) 9 (1) Double NTD 29 (3) 0 6 (1 ) 35 (4) Total 930 (94) 15 (1.5) 46 (4.5) 99 1

"Percentage of all cases. bCL, cleft lip; CP, cleft palate; MCA, multiple congenital abnormalities.

due to additional information from hospital records. Six additional cases of NTDs with major malformations not originally found on a birth record were also ascertained in this manner. Of the 61 cases (6%) with other major malformations, 15 had cleft lip and/or cleft palate only, while 4 others had diagnoses of known syndromes (Apert syndrome with encephalocele, achondroplasia with spina bifida, and two cases of Down syndrome with spina bifida). Two additional cases with known diagnoses (Apert syndrome with encephalocele, trisomy 18 with spina bifida) were born in Utah to nonresidents. They are not included in our analyses. Table I11 lists the malforma- tions identified in the other 42 cases. The table divides the cases into those having only midline defects and those considered to have nonmidline defects or defects thought to represent a broader pattern of malformation.

Analyses NTD prevalence was 1.01 per lo00 live births or 1.00 per lo00 recorded

deliveries (Table IV). The male/female sex ratio for isolated NTDs is 0.672 and it is 0.966 for cases with other major malformations. The difference between the percent- ages of males in these two categories (40.2 versus 49%, respectively) is not significant (p > 0.1 ; arcsine test for the equality of two percentages). The nonsignificance in this case is probably due to the small denominator (57 of known sex) in the group of NTDs with other major malformations. Further epidemiological analyses of NTD cases with major malformations were uninformative due to low numbers. However, such analyses have been completed for the isolated NTD cases (eg, time-space clustering, long term trends, and seasonality, etc.) [Jorde et al, 19831.

522 Martin, Fineman, and Jorde

TABLE 111. Cases of NTDs With Other Conxenital Anomalies (N = 42*)

Nonmidline defects or defects indicative of a complex syndrome

NTD type Midline defects (22 cases) (20 cases)

Anencephaly 2 Omphalocele 1 Midanterior wall evisceration (thoracic

and abdominal)

Encephalocele 1 Imperforate anus 1 Congenital heart disease

Spina bifida 2 Imperforate anus 2 Imperforate anus with abnormal or ab-

sent genitalia 1 Omphalocele 1 Omphalocele with kidney and bowel

obstruction 1 Omphalocele with extrophy of bladder (?

OEIS) 1 Midline teratoma on buttocks 2 T-Efistula 1 Cleft lipicleft palate, microcephaly 1 Cleft palate, congenital heart disease 2 Congenital heart disease

Anencephaly with 1 Midanterior wall evisceration (thoracic) spina bifida 1 Omphalocele

Encephalocele with spina bifida

1 Skull adherent to placenta (? amniotic bands)

1 Polycystic kidney, accessory spleen (? Meckel syndrome)

1 Bilateral cleft liplcleft palate, bilateral colobomas

2 Cyclopia (? trisomy 13) 2 Syndactyly, cleft palate

1 Bilateral polydactyly, dextro- cardia, absent eyes and nose (? trisomy 13)

kidney 1 Bilateral Wilms’ tumor of

1 “Skeletal defects” 1 Syndactyly 1 Syndactyly, atresia of genitalia 1 Syndactyly, missing thumb, ab-

1 Transverse hemimelia, imper-

1 Absent ears 1 Absent ears, single nostril,

1 Absent ear canals, congenital

sent eyes and nose

forate anus with a persistent tail

cleft lip/cleft palate

heart disease, cleft palate

1 Absent arms and legs 1 Bilateral cleft lip, polydactyly

1 Transverse hemimelia (leg), (? trisomy 13)

large abdomen

1 Craniosynostosis

Craniorachischisis 1 Omphalocele, undeveloped genitalia

*This does not include known syndromes (4), cases with isolated cleft lip and/or cleft palate (15), or anomalies which would be considered secondary to NTD lesion.

TABLE IV. Prevalence and Sex Ratio

Prevalence at birth 1.00/1000 recorded deliveries (including fetal deaths) 1.0111OOO live births)”

Isolated (923 cases)a With other malformations (57 cases)a

Sex Ratio a / Q Anancephaly 0.535 0.857 Spina Bifida 0.776 1.000 Encephalocele 0.722 1.000 Overall 0.672 0.996

aseven isolated cases and four cases with MCA were of unknown sex

Neural Tube Defect Heterogeneity 523

Based on a prevalence at birth (including stillborns) of 1/1OOO for NTDs and 1.64/1OOO Woolf et al, 19631 for cleft lip and/or cleft palate in Utah, the expected number of cases in our population having both malformations would be two, assuming their statistical independence. In fact, there were 15 cases that had both malforma- tions. Based on these figures, the conditional probability that a case has cleft lip/ palate given that it has an NTD is 0.0151, while the conditional probability that a case has cleft liplpalate given that it does not have an NTD is only 0.0016. Since our enumeration of the population is virtually complete, these are exact probabilities with no sampling error.

There were 6 of 37 (17.1 %) cases with double NTDs that had additional major malformations, while 40 of 956 (4.3%) cases of single NTDs had additional malfor- mations. The difference between these two figures is significant (p < 0.001; arcsine test for the equality of two percentages).

Informative families of 187 cases with isolated NTDs linked into the Utah Genealogical Data Base; 285 sibs were born subsequently to these propositi. Nine of these sibs had an isolated NTD, yielding an isolated NTD recurrence risk of 3.2 % . Only nine informative families of cases of NTDs with other major malformations linked to the Data Base. In these families there were 16 subsequent sibs, none having an NTD.

A significant difference exists between the percentage of syndromal NTD cases in our study (6%) and those given in both the Atlanta BDMP (12%) and MACDP (20%) in Khoury’s study [1982a]. The 95% confidence intervals are 4.75-7.76, 11.54-12.87, and 16.95-23.63, respectively.

DISCUSSION

Of the 991 NTD cases ascertained in this retrospective 40-year study, 6% were found to have other major malformations. In their 3-year prospective study of 27 newborn and stillborn infants with NTDs, Holmes et a1 [ 19761 found four cases (15 %) with additional major malformations. Personal communication with Dr. Holmes, who now has accumulated 3 additional years of prospective data, indicates that this 15% figure still holds true although it does not include two cases of NTDs with chromo- some abnormalities, one case of encephalocele with osteogenesis imperfecta and one case of amnion-rupture sequence. Inclusion of these cases would suggest that approx- imately 20% of their cases with NTDs have other malformations beyond a secondary effect of the primary lesion. The retrospective data of Holmes et al (published in the same article) indicated that 46% (36/79) of cases with NTDs have additional major malformations. The difference between these two percentages is likely due to ascer- tainment bias since the retrospective data were derived from autopsy descriptions of affected infants. Besides the increased probability of finding a malformation at autopsy, NTD patients with other major malformations are also more likely to die and have an autopsy than those with an isolated NTD. Chance et a1 [1981] examined 73 anencephalics sent to them by various individuals in the Pacific Northwest and found that 32 % had additional major malformations. This figure would also be considered biased assuming unusual cases are more likely to be sent for examination. Recently, Khoury et a1 [1982a] published retrospective data from the CDC nationwide and Atlanta surveillance systems. They found that 12% and 20%, respectively, of the NTD cases had other major malformations. Our 6% figure is significantly lower

524 Martin, Fineman, and Jorde

(statistically) than the figures reported in these previous studies. Whether this differ- ence is real or artifactual cannot be determined. Vital statistics records are often incomplete [Mackeprang et al, 19721 and it is quite possible that 6% is an underesti- mate. On the other hand, we did locate hospital/physician charts on at least 200 cases first identified on birth or fetal death certificates and only 7 (about 3.5%) offered more information than that found on the corresponding vital statistics records. Extrap- olation of this 3.5% figure to the other 600 or so cases of presumed isolated NTDs (from birth and fetal death certificates) would only yield approximately 20 more cases. Addition of these cases to our population of NTDs with malformations would not move our 6% figure within the 95% confidence intervals of Khoury’s results. One other explanation for the discrepancy in these figures is the possibility that in the previously mentioned studies there was underascertainment of isolated NTD patients resulting in spuriously low denominators. Underascertainment of NTD cases is not considered a major problem in this study due to our multiple sources of ascertainment and consistent patterns of referral.

This study and others previously mentioned suggest that a significant proportion of individuals born with an NTD have conditions that are causally different from the isolated NTDs. This heterogeneity is suggested by the phenotypes presented in Table 111. The discrepancy in the male/female sex ratios between isolated NTDs (0.672) and those with major malformations (0.966) also identifies an important difference between the two groups. While this sex ratio difference in our study is not significant (most likely due to the small number of cases with major malformations) Khoury et al [1982a] found similar differences in larger surveys. Lastly, the difference in recurrence risk between the two groups (3.2 % versus 0%) suggests heterogeneity. However, this comparison must be viewed cautiously since the number of subsequent sibs (16) born to families of the NTD group with malformations is too small to show statistical significance. Khoury et a1 [1982b] found a 0% recurrence risk for their “multiple” cases but their numbers were also small. This lack of recurrence is consistent with other empiric risk data for unknown malformation syndromes [Bartley and Hall, 19781, but one should keep in mind that we did not examine these NTD patients. It is probable, when considering the various anomalies listed in Table 111, that some of these cases were the result of chromosomal or single-gene conditions with specific recurrence risks.

We observed an increased incidence in the concurrence of NTDs with oral clefts. This observation has been reported previously Warkany et al, 1971, Gorlin et al, 1976, Lemire et al, 19781. More recently, Czeizel [1981] found that omphalocele and diaphragmatic hernias as well as oral clefts seem to associate more frequently with NTDs. To explain these associations, Opitz and Gilbert [1982] have hypothesized that the midline is a developmental field having “poorly buffered” morphogenetic properties that results in “generalized midline weakness. ” Table I11 was divided, as objectively as possible, into two categories with this concept in mind. Although the Table lists many midline defects associated with NTDs, we chose only to analyze statistically the association of NTDs with oral clefts due to their relative ease of diagnosis. Not having personally examined any of the cases, we can only presume that the correct description or diagnosis was given. Because we did not see the cases listed in Table 111, we thought no other statistical correlations could be performed accurately with respect to the midline although about two-thirds of the cases with major malformations (if one includes the 15 with oral clefts not listed) seem to have

Neural Tube Defect Heterogeneity 525

just midline defects. It is possible some of these cases have other unrecognized or unreported anomalies as part of a broader pattern of malformation.

Our analysis also indicated that patients with double NTDs in this population seemed to have major malformations more often than those with single NTDs. The reason for this association is unknown and, to our knowledge, has not been reported before. Its actual significance is hard to evaluate due to the small numbers involved.

In summary, we conclude that phenotypic and probable causal heterogeneity exists in NTD patients. This fact should be considered with respect to diagnosis and genetic counseling. We also conclude that evidence for midline schisis association, especially in regard to the presence of cleft lip and/or cleft palate, is strong and should be examined further.

ACKNOWLEDGMENTS

Thanks are due to Debbie Russell for her secretarial help and to the Utah Bureau of Vital Statistics for their cooperation in data collection. We are also grateful to John Carey for his clinical insight and to Douglas Anderton for his aid in statistical analysis. This research was funded by a clinical research grant (grant #6-291) from the March of Dimes Birth Defects Foundation.

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Edited by John M. Opitz