The human embryonic genome is karyotypically complex, with chromosomally abnormal cells preferentially located away from the developing fetus.

STUDY QUESTION: Are chromosome abnormalities detected at Day 3 post-fertilization predominantly retained in structures of the blastocyst other than the inner cell mass (ICM), where chromosomally normal cells are preferentially retained? SUMMARY ANSWER: In human embryos, aneuploid cells are sequestered away from the ICM, partly to the trophectoderm (TE) but more significantly to the blastocoel fluid within the blastocoel cavity (Bc) and to peripheral cells (PCs) surrounding the blastocyst during Day 3 to Day 5 progression. WHAT IS KNOWN ALREADY: A commonly held dogma in all diploid eukaryotes is that two gametes, each with 'n' chromosomes (23 in humans), fuse to form a '2n' zygote (46 in humans); a state that remains in perpetuity for all somatic cell divisions. Human embryos, however, display high levels of chromosomal aneuploidy in early stages that reportedly declines from Day 3 (cleavage stage) to Day 5 (blastocyst) post-fertilization. While this observation may be partly because of aneuploid embryonic arrest before blastulation, it could also be due to embryo 'normalization' to a euploid state during blastulation. If and how this normalization occurs requires further investigation. STUDY DESIGN, SIZE, DURATION: A total of 964 cleavage-stage (Day 3) embryos underwent single-cell biopsy and diagnosis for chromosome constitution. All were maintained in culture, assessing blastulation rate, both for those assessed euploid and aneuploid. Pregnancy rate was assessed for those determined euploid, blastulated and subsequently transferred. For those determined aneuploid and blastulated (174 embryos), ICM (all 174 embryos), TE (all 174), Bc (47 embryos) and PC (38 embryos) were analyzed for chromosome constitution. Specifically, concordance with the original Day 3 diagnosis and determination if any 'normalized' to euploid karyotypes within all four structures was assessed. PARTICIPANTS/MATERIALS, SETTING, METHODS: All patients (144 couples) were undergoing routine preimplantation genetic testing for aneuploidy in three IVF clinical settings. Cleavage-stage biopsy preceded chromosome analysis by next-generation sequencing. All patients provided informed consent. Additional molecular testing was carried out on blastocyst embryos and was analyzed for up to four embryonic structures (ICM, TE, Bc and PC). MAIN RESULTS AND THE ROLE OF CHANCE: Of 463/964 embryos (48%) diagnosed as euploid at Day 3, 70% blastulated (leading to a 59% pregnancy rate) and 30% degenerated. Conversely, of the 501 (52%) diagnosed as aneuploid, 65% degenerated and 35% (174) blastulated, a highly significant difference (P < 0.0001). Of the 174 that blastulated, the ratio of '(semi)concordant-aneuploid' versus 'normalized-euploid' versus 'other-aneuploid' embryos was, respectively, 39%/57%/3% in the ICM; 49%/48%/3% in the TE; 78%/21%/0% in the PC; and 83%/10%/5% in the Bc. The TE karyotype therefore has a positive predictive value of 86.7% in determining that of the ICM, albeit with marginally higher aneuploid rates of abnormalities (P = .071). Levels of abnormality in Bc/PC were significantly higher (P < 0.0001) versus the ploidy of the ICM and TE and nearly all chromosome abnormalities were (at least partially) concordant with Day 3 diagnoses. LIMITATIONS, REASONS FOR CAUTION: The results only pertain to human IVF embryos so extrapolation to the in vivo situation and to other species is not certain. We acknowledge (rather than lineage-specific survival, as we suggest here) the possibility of other mechanisms, such as lineage-specific movement of cells, during blastulation. Ethical considerations, however, make investigating this mechanism difficult on human embryos. WIDER IMPLICATIONS OF THE FINDINGS: Mosaic human cleavage-stage embryos can differentiate into a euploid ICM where euploid cell populations predominate. Sequestering of aneuploid cells/nuclei to structures no longer involved in fetal development has important implications for preimplantation and prenatal genetic testing. These results also challenge previous fundamental understandings of mitotic fidelity in early human development and indicate a complex and fluid nature of the human embryonic genome. STUDY FUNDING/COMPETING INTEREST(S): This research was funded by Organon Pharmaceuticals and Merck Serono by grants to W.G.K. W.G.K. is also an employee of AdvaGenix, who could, potentially, indirectly benefit financially from publication of this manuscript. R.C.M. is supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number R35GM133747. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. D.K.G. provides paid consultancy services for Care Fertility. TRIAL REGISTRATION NUMBER: : N/A.

Genetic basis of variable exon 9 skipping in cystic fibrosis transmembrane conductance regulator mRNA.

Variable in-frame skipping of exon 9 in cystic fibrosis transmembrane conductance regulator (CFTR) mRNA transcripts (exon 9-) occurs in the respiratory epithelium. To explore the genetic basis of this event, we evaluated respiratory epithelial cells and blood leukocytes from 124 individuals (38 with cystic fibrosis (CF), 86 without CF). We found an inverse relationship between the length of the polythymidine tract at the exon 9 splice branch/acceptor site and the proportion of exon 9- CFTR mRNA transcripts. These results strongly indicate a genetic basis in vivo modulating post-transcriptional processing of CFTR mRNA transcripts.

ECFS standards of care on CFTR-related disorders: Diagnostic criteria of CFTR dysfunction.

The spectrum of disorders involving CFTR (cystic fibrosis transmembrane conductance regulator) dysfunction correlates with a continuous gradient of CFTR function defined by the combination of two allelic CFTR variants. CFTR-related disorders are clinical entities with features of cystic fibrosis (CF) and evidence for presence of CFTR dysfunction but not meeting criteria for diagnosis of CF. Individuals with CFTR-RDs demonstrate a wide range of CFTR activity and are still under-recognized or misclassified. The level of CFTR dysfunction may be measured in vivo (sweat testing, nasal potential difference measurements) and/or by ex vivo tests (intestinal current measurement), or indirectly indicated by CFTR variants, as alteration in sequence of the CFTR gene translates into CFTR dysfunction. CFTR bioassays can aid in the diagnosis of individuals with CF, but we lack parameters to differentiate CF from CFTR-RD. In the era of the CFTR modulators and their potential clinical benefit, it is of utmost importance to diagnose CFTR-RD as unambiguously as possible. We therefore propose the following to define compatible CFTR dysfunction in a person with a suspected diagnosis of CFTR-RD : (1) evidence of CFTR dysfunction in vivo or ex vivo in at least two different CFTR functional test types, or (2) One CFTR variant known to reduce CFTR function and evidence of CFTR dysfunction in vivo or ex vivo in at least two different CFTR functional test types, or (3) Two CFTR variants shown to reduce CFTR function, with at most one CF-causing variant.

Understanding the population structure of North American patients with cystic fibrosis.

It is generally presumed that the cystic fibrosis (CF) population is relatively homogeneous, and predominantly of European origin. The complex ethnic make-up observed in the CF patients collected by the North American CF Modifier Gene Consortium has brought this assumption into question, and suggested the potential for population substructure in the three CF study samples collected from North America. It is well appreciated that population substructure can result in spurious genetic associations. To understand the ethnic composition of the North American CF population, and to assess the need for population structure adjustment in genetic association studies with North American CF patients, genome-wide single-nucleotide polymorphisms on 3076 unrelated North American CF patients were used to perform population structure analyses. We compared self-reported ethnicity to genotype-inferred ancestry, and also examined whether geographic distribution and cystic fibrosis transmembrane regulator (CFTR) mutation type could explain the population structure observed. Although largely Caucasian, our analyses identified a considerable number of CF patients with admixed African-Caucasian, Mexican-Caucasian and Indian-Caucasian ancestries. Population substructure was present and comparable across the three studies of the consortium. Neither geographic distribution nor CFTR mutation type explained the population structure. Given the ethnic diversity of the North American CF population, it is essential to carefully detect, estimate and adjust for population substructure to guard against potential spurious findings in CF genetic association studies. Other Mendelian diseases that are presumed to predominantly affect single ethnic groups may also benefit from careful analysis of population structure.

The genetics and genomics of cystic fibrosis.

Genetics is the branch of biology concerned with study of individual genes and how they work whereas genomics is involved with the analysis of all genes and their interactions. Both of these approaches have been applied extensively to CF. Identification of the CFTR gene initiated the dissection of CF genetics at the molecular level. Subsequently, thousands of variants were found in the gene and the functional consequences of a subset have been studied in detail. The completion of the human genome ushered in a new phase of study where the role of genes beyond CFTR could be evaluated for their contribution to the severity of CF. This will be a brief overview of the contribution of these complementary methods to our understanding of CF pathogenesis.

A susceptibility gene for type 2 diabetes confers substantial risk for diabetes complicating cystic fibrosis.

AIMS/HYPOTHESIS: Insulin-requiring diabetes affects 25-50% of young adults with cystic fibrosis (CF). Although the cause of diabetes in CF is unknown, recent heritability studies in CF twins and siblings indicate that genetic modifiers play a substantial role. We sought to assess whether genes conferring risk for diabetes in the general population may play a risk modifying role in CF. METHODS: We tested whether a family history of type 2 diabetes affected diabetes risk in CF patients in 539 families in the CF Twin and Sibling family-based study. A type 2 diabetes susceptibility gene (transcription factor 7-like 2, or TCF7L2) was evaluated for association with diabetes in CF using 998 patients from the family-based study and 802 unrelated CF patients in an independent case-control study. RESULTS: Family history of type 2 diabetes increased the risk of diabetes in CF (OR 3.1; p = 0.0009). A variant in TCF7L2 associated with type 2 diabetes (the T allele at rs7903146) was associated with diabetes in CF in the family study (p = 0.004) and in the case-control study (p = 0.02; combined p = 0.0002). In the family-based study, variation in TCF7L2 increased the risk of diabetes about three-fold (HR 1.75 per allele, 95% CI 1.3-2.4; p = 0.0006), and decreased the mean age at diabetes diagnosis by 7 years. In CF patients not treated with systemic glucocorticoids, the effect of TCF7L2 was even greater (HR 2.9 per allele, 95% CI 1.7-4.9, p = 0.00011). CONCLUSIONS/INTERPRETATION: A genetic variant conferring risk for type 2 diabetes in the general population is a modifier of risk for diabetes in CF.

A synonymous mutation in TCOF1 causes Treacher Collins syndrome due to mis-splicing of a constitutive exon.

Interpretation of the pathogenicity of sequence alterations in disease-associated genes is challenging. This is especially true for novel alterations that lack obvious functional consequences. We report here on a patient with Treacher Collins syndrome (TCS) found to carry a previously reported mutation, c.122C > T, which predicts p.A41V, and a novel synonymous mutation, c.3612A > C. Pedigree analysis showed that the c.122C > T mutation segregated with normal phenotypes in multiple family members while the c.3612A > C was de novo in the patient. Analysis of TCOF1 RNA in lymphocytes showed a transcript missing exon 22. These results show that TCS in the patient is due to haploinsufficiency of TCOF1 caused by the synonymous de novo c.3612A > C mutation. This study highlights the importance of clinical and pedigree evaluation in the interpretation of known and novel sequence alterations.

The skipping of constitutive exons in vivo induced by nonsense mutations.

Nonsense mutations create a premature signal for the termination of translation of messenger RNA. Such mutations have been observed to cause a severe reduction in the amount of mutant allele transcript or to generate a peptide truncated at the carboxyl end. Analysis of fibrillin transcript from a patient with Marfan syndrome revealed the skipping of a constitutive exon containing a nonsense mutation. Similar results were observed for two nonsense mutations in the gene encoding ornithine delta-aminotransferase from patients with gyrate atrophy. All genomic DNA sequences flanking these exons that are known to influence RNA splicing were unaltered, which suggests that nonsense mutations can alter splice site selection in vivo.

Decreased mRNA and protein stability of W1282X limits response to modulator therapy.

BACKGROUND: Cell-based studies have shown that W1282X generates a truncated protein that can be functionally augmented by modulators. However, modulator treatment of primary cells from individuals who carry two copies of W1282X generates no functional CFTR. To understand the lack of response to modulators, we investigated the effect of W1282X on CFTR RNA transcript levels. METHODS: qRT-PCR and RNA-seq were performed on primary nasal epithelial (NE) cells of a previously studied individual who is homozygous for W1282X, her carrier parents and control individuals without nonsense variants in CFTR. RESULTS: CFTR RNA bearing W1282X in NE cells shows a steady-state level of 4.2 ±â€¯0.9% of wild-type (WT) CFTR RNA in the mother and 12.4 ±â€¯1.3% in the father. NMDI14, an inhibitor of nonsense-mediated mRNA decay (NMD), restored W1282X mRNA to almost 50% of WT levels in the parental NE cells. RNA-seq of the NE cells homozygous for W1282X showed that CFTR transcript level was reduced to 1.7% of WT (p-value: 4.6e-3). Negligible truncated CFTR protein was generated by Flp-In 293 cells stably expressing the W1282X EMG even though CFTR transcript was well above levels observed in the parents and proband. Finally, we demonstrated that NMD inhibition improved the stability and response to correctors of W1282X-CFTR protein expressed in the Flp-In-293 cells. CONCLUSION: These results show that W1282X can cause substantial degradation of CFTR mRNA that has to be addressed before efforts aimed at augmenting CFTR protein function can be effective.

Consensus on the use and interpretation of cystic fibrosis mutation analysis in clinical practice.

It is often challenging for the clinician interested in cystic fibrosis (CF) to interpret molecular genetic results, and to integrate them in the diagnostic process. The limitations of genotyping technology, the choice of mutations to be tested, and the clinical context in which the test is administered can all influence how genetic information is interpreted. This paper describes the conclusions of a consensus conference to address the use and interpretation of CF mutation analysis in clinical settings. Although the diagnosis of CF is usually straightforward, care needs to be exercised in the use and interpretation of genetic tests: genotype information is not the final arbiter of a clinical diagnosis of CF or CF transmembrane conductance regulator (CFTR) protein related disorders. The diagnosis of these conditions is primarily based on the clinical presentation, and is supported by evaluation of CFTR function (sweat testing, nasal potential difference) and genetic analysis. None of these features are sufficient on their own to make a diagnosis of CF or CFTR-related disorders. Broad genotype/phenotype associations are useful in epidemiological studies, but CFTR genotype does not accurately predict individual outcome. The use of CFTR genotype for prediction of prognosis in people with CF at the time of their diagnosis is not recommended. The importance of communication between clinicians and medical genetic laboratories is emphasized. The results of testing and their implications should be reported in a manner understandable to the clinicians caring for CF patients.

Extensive posttranscriptional deletion of the coding sequences for part of nucleotide-binding fold 1 in respiratory epithelial mRNA transcripts of the cystic fibrosis transmembrane conductance regulator gene is not associated with the clinical manifestations of cystic fibrosis.

Cystic fibrosis (CF) is a recessive hereditary disorder, requiring both parental cystic fibrosis conductance transmembrane regulator (CFTR) genes to carry mutations for clinical disease to manifest, i.e., only 50% of normal CFTR gene expression is required to maintain a normal phenotype. To help define the minimum amount of normal CFTR gene expression necessary to maintain normalcy, we have capitalized on our prior observation (Chu, C.-S., B. C. Trapnell, J. J. Murtagh, Jr., J. Moss, W. Dalemans, S. Jallat, A. Mercenier, A. Pavirani, J.-P. Lecocq, G. R. Cutting, et al. 1991. EMBO [Eur. Mol. Biol. Organ] J. 10:1355-1363) that normal individuals can have up to 66% of bronchial CFTR mRNA transcripts that are missing exon 9, a region representing 21% of the sequence coding for the critical nucleotide (ATP)-binding fold 1 (NBF1) of the predicted CFTR protein. The study population included 78 individuals with no prior diagnosis of CF. Evaluation of bronchial epithelial cells (obtained by bronchoscopy) revealed that exon 9 was variably deleted in all individuals. Remarkably, there were four individuals, all greater than or equal to 35 yr, in whom bronchial epithelial cells exhibited 73, 89, 90, and 92% CFTR transcripts with inframe deletion of exon 9, respectively, despite normal sweat Cl- and no clinical manifestation of CF. In the context that only 8% or less of bronchial CFTR transcripts need exon 9 to maintain normal airway function, these observations strongly suggest that either exon 9 is not necessary for CFTR structure and/or function or that only a very small fraction of bronchial epithelial cells need to express normal CFTR mRNA transcripts with exon 9 to perform the function of CFTR sufficient to maintain a normal phenotype in vivo.

Marfan phenotype variability in a family segregating a missense mutation in the epidermal growth factor-like motif of the fibrillin gene.

To examine the associations among fibrillin gene mutations, protein function, and Marfan syndrome phenotype, we screened for alterations in the fibrillin coding sequence in patients with a range of manifestations and clinical severity. A cysteine to serine substitution at codon 1409 (C1409S) was identified in an epidermal growth factor (EGF)-like motif from one fibrillin allele which segregates with the disease phenotype through three generations of a family affected with the Marfan syndrome. This alteration was not observed in 60 probands from other families or in 88 unrelated normal individuals. The altered cysteine is completely conserved in all EGF-like motifs identified in fibrillin, and in all proteins that contain this motif. These observations strongly indicate that C1409S is the disease-producing mutation in this family. The phenotype of individuals carrying C1409S varied widely with respect to onset of disease, organ-system involvement, and clinical severity; certain affected adults were unaware of their status before being diagnosed through this investigation. We conclude that fibrillin gene defects cause familial Marfan syndrome, that mutations in the EGF-like motif of the fibrillin gene are not uniformly associated with severe disease, and that fibrillin genotype is not the sole determinant of Marfan phenotype.

Severe deficiency of cystic fibrosis transmembrane conductance regulator messenger RNA carrying nonsense mutations R553X and W1316X in respiratory epithelial cells of patients with cystic fibrosis.

Cystic fibrosis (CF) is the most common, lethal inherited disorder in the Caucasian population. We have recently reported two African-American patients with nonsense mutations in each CF gene and severe pancreatic disease, but mild pulmonary disease. In order to examine the effect of these nonsense mutations on CF gene expression, bronchial and nasal epithelial cells were obtained from one of these patients (no. 246), a compound heterozygote for nonsense mutations R553X and W1316X; a healthy normal individual; a patient (no. 528) homozygous for the common CF mutation (delta F508); and a CF patient (no. 272) who carries the R553X mutation and a missense mutation, S549N. When mRNA from bronchial cells of the normal individual, the delta F508 homozygote, and the S549N/R553X compound heterozygote was reverse transcribed and amplified by polymerase chain reaction using primers derived from the CF gene, DNA fragments of the predicted size were observed. However, patient no. 246 with nonsense mutations in each CF gene has no detectable cystic fibrosis transmembrane conductance regulator (CFTR) messenger RNA, and therefore should have severely diminished, and possibly absent, CFTR protein. Furthermore, less than 2% of the CFTR transcripts in nasal epithelial cells from patient no. 272 (S549N/R553X) were derived from the gene with the nonsense mutation. We conclude that severe reduction in CFTR mRNA causes CF, but can have different consequences in the lung and pancreas.

A PDZ-interacting domain in CFTR is an apical membrane polarization signal.

Polarization of the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel, to the apical plasma membrane of epithelial cells is critical for vectorial transport of chloride in a variety of epithelia, including the airway, pancreas, intestine, and kidney. However, the motifs that localize CFTR to the apical membrane are unknown. We report that the last 3 amino acids in the COOH-terminus of CFTR (T-R-L) comprise a PDZ-interacting domain that is required for the polarization of CFTR to the apical plasma membrane in human airway and kidney epithelial cells. In addition, the CFTR mutant, S1455X, which lacks the 26 COOH-terminal amino acids, including the PDZ-interacting domain, is mispolarized to the lateral membrane. We also demonstrate that CFTR binds to ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50), an apical membrane PDZ domain-containing protein. We propose that COOH-terminal deletions of CFTR, which represent about 10% of CFTR mutations, result in defective vectorial chloride transport, partly by altering the polarized distribution of CFTR in epithelial cells. Moreover, our data demonstrate that PDZ-interacting domains and PDZ domain-containing proteins play a key role in the apical polarization of ion channels in epithelial cells.

Expression of the voltage-gated chloride channel ClC-2 in rod bipolar cells of the rat retina.

Voltage-gated chloride channels (ClC) are highly conserved during evolution and appear to participate in a variety of physiological functions. Recently, ClC-2 was proposed to play a role in stabilizing the chloride equilibrium potential near or below the resting membrane potential in neurons expressing ligand-gated chloride channels. Because rod bipolar cells in mammalian retina express three forms of inhibitory ligand-gated chloride channels, we decided to study ClC-2 localization and function in the rat retina. RNA encoding ClC-1, -2, -3, -4, and -5 was detected by reverse transcription-PCR in the rat retina. ClC-2-specific antibodies identified protein on cell bodies and in synaptic layers. Double-immunofluorescence staining revealed that intense ClC-2 immunoreactivity colocalized with PKC-stained rod bipolar cells. Patch-clamp experiments performed with individual rod bipolar cells demonstrated the presence of a time-dependent, inwardly rectified current activated at hyperpolarizing membrane potentials. This current demonstrated selectivity for different anions (Cl(-) > I(-) > gluconate), was inhibited by Cd(2+), and was minimally reduced by 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid. These features are consistent with currents generated by ClC-2 channels. Our data indicate that functional ClC-2 channels are present in retinal rod bipolar cells and support a role for ClC-2 in maintaining Cl(-) homeostasis in neurons with ligand-gated chloride channels.

ABCD1 mutations and the X-linked adrenoleukodystrophy mutation database: role in diagnosis and clinical correlations.

X-linked adrenoleukodystrophy (X-ALD) is caused by mutations in the ABCD1 gene, which encodes a peroxisomal ABC half-transporter (ALDP) involved in the import of very long-chain fatty acids (VLCFA) into the peroxisome. The disease is characterized by a striking and unpredictable variation in phenotypic expression. Phenotypes include the rapidly progressive childhood cerebral form (CCALD), the milder adult form, adrenomyeloneuropathy (AMN), and variants without neurologic involvement. There is no apparent correlation between genotype and phenotype. In males, unambiguous diagnosis can be achieved by demonstration of elevated levels of VLCFA in plasma. In 15 to 20% of obligate heterozygotes, however, test results are false-negative. Therefore, mutation analysis is the only reliable method for the identification of heterozygotes. Since most X-ALD kindreds have a unique mutation, a great number of mutations have been identified in the ABCD1 gene in the last seven years. In order to catalog and facilitate the analysis of these mutations, we have established a mutation database for X-ALD ( http://www.x-ald.nl). In this review we report a detailed analysis of all 406 X-ALD mutations currently included in the database. Also, we present 47 novel mutations. In addition, we review the various X-ALD phenotypes, the different diagnostic tools, and the need for extended family screening for the identification of new patients.

A 100 amino acid region in the GABA rho 1 subunit confers robust homo-oligomeric expression.

Retinal gamma-aminobutyric acid type C (GABAC) receptors are believed to be composed of rho subunits. Although rho 1 and rho 2 are over 80% similar, the whole-cell currents generated by rho 1 receptors in Xenopus oocytes are significantly greater than those generated by rho 2 receptors. In this study, chimeric subunits containing different portions of human rho 1 and human rho 2 were created to localize sequences facilitating robust rho 1 expression. Our results indicate that these sequences reside in a 100 amino acid domain in the N-terminus of rho 1, and may involve N-linked glycosylation. Since the N-terminus also contains subunit assembly signals, rho 1 receptors may be formed more efficiently than rho 2 receptors. Therefore, this study furthers our understanding of the molecular basis of GABA-mediated inhibition in the retina.

Identification of 70 amino acids important for GABA(C) receptor rho1 subunit assembly.

gamma-Aminobutyric acid (GABA) is the most important inhibitory neurotransmitter in the mammalian central nervous system and gates at least three subclasses of receptors, termed GABA(A), GABA(B) and GABA(C). Accumulating evidence indicates that GABA(C) receptors are composed exclusively of rho subunits. The N-terminal half of the rho subunits has been shown to mediate formation of homo- and heterooligomeric GABA(C) receptors. In this study, we searched for specific sequences within the N-terminus of the rho1 subunit involved in the assembly process. Assembly sequences were localized to a 128-amino acid region by deletion of progressively larger regions of a chimeric rho1beta1 subunit previously shown to disrupt rho1 and rho2 assembly. To confirm this observation, a series of GABA(A) receptor beta subunit chimeras containing different regions of the rho1 N-terminus were tested for interference with rho1 and rho2 subunit assembly into functional GABA receptors. Transfer of 70 residues within the 128 amino acid region to the beta1 subunit created a chimera that disrupted rho1, but not rho2, assembly into functional receptors. These observations refine the location of signals involved in rho1 subunit assembly, and suggest that different signals exist for the formation of rho1 homooligomeric and rho1/rho2 heterooligomeric GABA(C) receptors.