Faculty Bibliography

Using a case-control design, an association of schizophrenia with the dopamine D3 receptor gene (D3RG) locus was investigated. Initial analysis of pooled results from published studies revealed a significant excess of individuals homozygous for either allele among the patients. The association was next tested in two cohorts ascertained independently at Pittsburgh, Pennsylvania and at Houston, Texas. The Pittsburgh sample was comprised of patients with schizophrenia (DSM-III-R) (n = 130). The controls belonged to two groups: adults screened for the absence of substance abuse or major psychiatric illness (n = 128), and neonates (n = 160). Multivariate analysis suggested an association with allele 1 of the biallelic D3RG polymorphism in comparison with the adult, but not the neonatal, controls. The association was most marked among Caucasian patients with a family history of schizophrenia (odds ratio 13.69, confidence intervals 1.80, 104.30). Survival analysis suggested an earlier age of onset among male patients homozygous for allele 2. The Houston cohort included Caucasian patients with schizophrenia or schizoaffective disorder (DSM-III-R criteria, n = 50), and normal controls matched for gender (n = 51). In this group, no significant associations were noted among all the patients or among subgroups of patients based on family history or age of onset. Possible reasons for the discordant results are discussed.

Hirschsprung disease (HSCR), or congenital aganglionic megacolon, is the most common cause of congenital bowel obstruction with an incidence of 1 in 5000 live births. HSCR may be inherited as a single gene disorder with reduced penetrance or as a multigenic trait. HSCR mutations have been identified in the RET receptor tyrosine kinase, endothelin-B receptor (EDNRB) and its physiological ligand, endothelin 3 (EDN3). Although RET's ligand has remained elusive, it is expected to be an extracellular neurotrophic molecule expressed in the developing gut and kidney mesenchyme, based on the phenotypes of intestinal aganglionosis and renal agenesis observed in homozygous RET knockout (Ret -/-) mice. The glial cell line-derived neurotrophic factor (GDNF) is such a molecule. Recently, mice carrying two null alleles for Gdnf were shown to exhibit phenotypes remarkably similar to Ret-/- animals. We screened 106 unrelated HSCR patients for mutations in GDNF by direct sequencing. We identified one familial mutation in a HSCR patient with a known de novo RET mutation and malrotation of the gut. No haplotype sharing was evident in any of 36 HSCR kindreds typed for microsatellite markers surrounding GDNF on human chromosome 5p. Our data suggest that GDNF is a minor contributor to human HSCR susceptibility and that loss of its function in enteric neurogenesis may be compensated for by other neurotrophic factors or via other pathways. However, it may be that in rare instances, RET and GDNF mutations act in concert to produce an enteric phenotype.

Microcephaly is a heterogeneous disorder with genetic and environmental causes. However, there is little information on what proportion of cases are caused by inherited susceptibility, or the mode of inheritance in familial cases. To address these questions, we have performed classical and complex segregation analyses for microcephaly on 2 sets of family data collected from genetic counseling clinics in Vancouver and Jerusalem. These samples consisted of 143 affected individuals in 127 families ascertained from Vancouver, and 101 affected individuals in 59 families ascertained from Jerusalem. The results of the segregation analyses for the Vancouver sample indicated that approximately half of all microcephaly cases were due to highly penetrant recessive mutant alleles, with the remainder being sporadic. Although a recessive model allowing for the occurrence of sporadic cases fit the data from Vancouver best, a dominant model could not be statistically rejected. The classical segregation analysis on the Jerusalem sample suggested that both a dominant model with 29% of the cases being sporadic and a purely recessive model provided adequate fit to the data. Although the complex segregation analysis of this sample indicated that a dominant model provided a more parsimonious explanation for the observed familial variation, a recessive model was only marginally rejected. It should be noted that in the Jerusalem sample, families tended to be ascertained in the genetic counseling clinic only after the birth of a second affected child. This could be a potential bias which could inflate the segregation ratio, thus giving the impression of dominant inheritance. Our analyses, while confirming the complex nature of the cause of microcephaly, indicate that it may be necessary to await the results of genetic linkage analysis before a definitive mode of inheritance can be determined.

The familial aggregation of rheumatoid arthritis was examined to determine factors modifying the risk of rheumatoid arthritis in first degree relatives of 165 cases ascertained from January 1, 1987, through March 31, 1987, using the Saint Margaret Memorial Hospital Rheumatoid Arthritis Registry, Pittsburgh, Pennsylvania, without regard to previous information concerning the occurrence of rheumatoid arthritis among their family members. The reported affection status of first degree relatives, verified through a structured clinical evaluation, revealed a false-positive reporting rate for family members of 61%. In contrast, there were no false-negative cases detected. There were no differences in average family size or total number of years at risk between 135 simplex and 30 multiplex families; however, aggregation analysis revealed that only 18 of 30 confirmed multiplex families had significant excess risk of rheumatoid arthritis. Significant differences were found when probands from multiplex families were compared with those from simplex families with regard to female to male ratio for probands (1:1 in multiplex families vs. 3:1 in simplex families) and average age of onset for probands (41 years in multiplex families vs. 48 years in simplex families). The familial risk for rheumatoid arthritis was similar in parents (4.2%) and siblings (4.6%) and lowest for children (0.7%) of probands. The authors assert that the affection status of first degree relatives of patients with rheumatoid arthritis is often falsely reported as positive. The familiality of rheumatoid arthritis may be more accurately related to the sex and age at onset of the affected family member.

In an effort to contribute to the transcript map of human chromosome 21 and the understanding of the pathophysiology of trisomy 21, we have used exon trapping to identify fragments of chromosome 21 genes. Two trapped exons, from pools of chromosome 21-specific cosmids, showed homology to the Drosophila white (w) gene. We subsequently cloned the corresponding cDNA for a human homologue of the Drosophila w gene (hW) from human retina and fetal brain cDNA libraries. The gene belongs to the ATP-binding cassette transporter gene family and is homologous to Drosophila w (and to w genes from other species) and to a lesser extent to Drosophila brown (bw) and scarlet (st) genes that are all involved in the transport of eye pigment precursor molecules. A DNA polymorphism with 62% heterozygosity due to variation of a poly (T) region in the 3' UTR of the hW has been identified and used for the incorporation of this gene to the genetic map of chromosome 21. The hW is located at 21q22.3 between DNA markers D21S212 and D21S49 in a P1 clone that also contains marker BCEI. The gene is expressed at various levels in many human tissues. The contributions of this gene to the Down syndrome phenotypes, to human eye color, and to the resulting phenotypes of null or missense mutations are presently unknown.

Congenital central hypoventilation syndrome (CCHS) usually occurs as an isolated phenotype. However, 16% of the index cases are also affected with Hirschsprung disease (HSCR). Complex segregation analysis suggests that CCHS is familial and has the same inheritance pattern with or without HSCR. We postulate that alteration of normal function of the receptor tyrosine kinase, RET, may contribute to CCHS based on RET's expression pattern and the identification of RET mutations in HSCR patients. To further explore the nature of the inheritance of CCHS, we have undertaken two main routes of investigation: cytogenetic analysis and mutation detection. Cytogenetic analysis of metaphase chromosomes showed normal karyotypes in 13 of the 14 evaluated index cases; one index case carried a familial pericentric inversion on chromosome 2. Mutation analysis showed no sequence changes unique to index cases, as compared to control individuals, and as studied by single strand conformational polymorphism (SSCP) analysis of the coding region of RET. We conclude that point mutations in the RET coding region cannot account for a substantial fraction of CCHS in this patient population, and that other candidate genes involved in neural crest cell differentiation and development must be considered.

Hirschsprung disease (HSCR) or colonic aganglionosis is a congenital disorder characterized by an absence of intramural ganglia along variable lengths of the colon resulting in intestinal obstruction. The incidence of HSCR is 1 in 5,000 live births. Mutations in the RET gene, which codes for a receptor tyrosine kinase, and in EDNRB which codes for the endothelin-B receptor, have been shown to be associated with HSCR in humans. The lethal-spotted mouse which has pigment abnormalities, but also colonic aganglionosis, carries a mutation in the gene coding for endothelin 3 (Edn3), the ligand for the receptor protein encoded by EDNRB. Here, we describe a mutation of the human gene for endothelin 3 (EDN3), homozygously present in a patient with a combined Waardenburg syndrome type 2 (WS2) and HSCR phenotype (Shah-Waardenburg syndrome). The mutation, Cys159Phe, in exon 3 in the ET-3 like domain of EDN3, presumably affects the proteolytic processing of the preproendothelin to the mature peptide EDN3. The patient's parents were first cousins. A previous child in this family had been diagnosed with a similar combination of HSCR, depigmentation and deafness. Depigmentation and deafness were present in other relatives. Moreover, we present a further indication for the involvement of EDNRB in HSCR by reporting a novel mutation detected in one of 40 unselected HSCR patients.