MATERIALS AND METHODS

Our population comprised 33 unrelated patients (31 from Germany, one from German Switzerland, and one from Austria) who were followed exclusively in specialised pulmonary care centres and met the criteria for CCHS, evaluated and diagnosed by a polygraphic respiratory recording which detected hypoventilation, high PaCO₂, and low PaO₂ during quiet sleep,³ and for whom the review of medical records yielded no further cause for the hypoventilation. None of them showed additional primary neuromuscular, pulmonary, or cardiac anomalies. Eight of the 33 patients (24.2%) had a concomitant HSCR phenotype. Hirschsprung disease was diagnosed by histological and histochemical evaluation of intestinal biopsies, confirming the absence of enteric ganglia and the increase in acetylcholinesterase in hyperplastic nerve fibres in the aganglionic tract.

One patient in our series had a monozygotic twin with the same clinical presentation of CCHS and another patient had an affected brother. All other patients were seemingly sporadic cases. Samples from previously described healthy German blood donors served as our normal, race and gender matched controls.³⁸ According to a questionnaire, they did not have any signs of congenital or tumoral disorders. Symptoms of ANS dysfunction were not evaluated. Our control population showed similar allele frequencies of RET polymorphisms compared to other unaffected European and North American populations. All studies were conducted with written informed consent from the patients and their families, and all protocols were approved by the Clinical Ethics Commission of the University of Dresden.

Genomic DNA was obtained from leucocytes derived from peripheral venous blood samples and isolated by standard protocols. The 21 RET exons were amplified from genomic DNA using primers and reaction conditions described by Ceccherini et al³⁹ for exons 1-4, 11, and 16, and by Mulligan et al⁴⁰ for exons 5, 7, 9, 10, 12, 13, 17, 18, and 20. To amplify the remaining exons we generated new primer pairs (exon 6: sense 5′-CAGAGCAGCTTGGTGGTCA-3′; antisense 5′-AGTC TACTCTGTGCTGGTTGG-3′; exon 8: sense 5′-GCACTAG CTGGACGCTGG-3′; antisense 5′-GAGACCATCCCAGGCTG GC-3′; exon 14: sense 5′-TGTGTCCACCCCCTTACTCATTGG-3′; antisense 5′-CGTGGTGGGTCAGGGTGTGG-3′; exon 15: sense 5′-CCCCCGGCCCAGGTCTCAC-3′; antisense 5′-GCTCCACTA ATCTTCGGTATCTTT-3′; exon 19: sense 5′-GCGGACATC AGCAAAGAC-3′; antisense 5′-GCTGGGCAATG TGGTAAT-3′; exon 21: sense 5′-CCGGGCCCACCACATCATC-3′; antisense 5′-AGCCCAAATTAGAGCCAGGTTACG-3′). Amplified DNA fragments were analysed by a direct DNA sequencing approach applying the Thermo Sequenase^™ Fluorescent Cycle Sequencing kit (Amersham Pharmacia Biotech, Freiburg, Germany) according to the manufacturer’s protocol. The sequencing primers were the same as the PCR primers with an additional Cy5 labelling, allowing sequence analysis on ALF Express^™ devices (Amersham Pharmacia Biotech, Freiburg, Germany).

We also investigated the genotype distribution of polymorphisms of positions c.135G/A, c.375C/A, c.1296G/A, c.2071G/A, c.2307T/G, c.2508C/T, and c.2712C/G of the coding region of the RET proto-oncogene. The seven investigated DNA fragments were amplified from genomic DNA using primers and methods described previously.³⁸ All analysed polymorphisms created or abolished a restriction site of an endonuclease, namely, EagI, MboII, BsmI, BanI, TaqI, AluI, and RsaI. Genotypes were determined by digestion of the PCR product and electrophoresis on a polyacrylamide gel. Statistical analysis was performed with the Pearson χ² test with a confidence interval of 95%.

RESULTS

Sequence analysis showed a RET germline mutation in a female patient with CCHS and central epilepsy, but without any symptoms of HSCR. The patient herself and the relatives investigated did not suffer from constipation, so we had no reason to evaluate the intestinal nervous system histologically. We found a nucleotide change (C>A) at position ds+54 of intron 12. This mutation was transmitted from the healthy father and we also detected this transversion in the asymptomatic brother.

Furthermore, we found a missense mutation of exon 13 codon 791 (TAT→TTT) in a male patient with CCHS in association with total colonic aganglionosis, which resulted in the substitution of a highly conserved tyrosine for phenylalanine. This Y791F mutation has been previously reported in a patient with sporadic HSCR,²⁸ as well as in a kindred with familial medullary thyroid carcinoma.⁴¹ In our patient, the mutation was inherited from the unaffected father and the family history for HSCR or MEN2 was negative. A third mutation was detected in exon 14 codon 841 (CCG→CTG) in a female CCHS/HSCR patient with short segment aganglionosis. This hitherto unknown C→T transition resulted in the substitution of a proline by a leucine. This mutation was not detected in the healthy mother, while genetic testing was not possible in the father. None of these three mutations was detected in 120 normal controls (table 1).

In addition to the mutations, we found a rare RET variant of exon 18 codon 982 (CGC→TGC)^42,43 in a male patient with combined CCHS and HSCR, which substituted a cysteine residue for an arginine. This variant was inherited from the healthy mother and was absent in 156 normal controls.

We have determined allele frequencies for seven RET polymorphisms in the CCHS population and compared the results to those in a normal control population. Only one of these seven polymorphisms showed a statistically significant association with the CCHS phenotype. The genotype distribution for each of the seven polymorphic loci did not deviate significantly from Hardy-Weinberg equilibrium.

A significant over-representation of the c.135A variant was found within the CCHS population in comparison to the controls (table 2, χ²=4.536, p=0.044). Furthermore, a similar tendency towards over-representation of the c.1296A variant of RET was found in the CCHS population, although the difference was not statistically significant (table 2, χ²=3.656, p=0.074). The allele frequencies of all other investigated polymorphisms, namely those of codons c.375C/A, c.2071G/A, c.2307T/G, c.2508C/T, and c.2712C/G, did not derive from that detected in our normal control population, which was matched for race and gender (table 2).

DISCUSSION

In this study, we investigated 25 patients with isolated CCHS and eight patients with CCHS in combination with HSCR for RET germline mutations in the 21 exons including the adjacent intronic sequences. We detected three germline mutations, one in a patient with a CCHS/epilepsy phenotype but no HSCR and two in patients with a CCHS/HSCR phenotype. Several studies have reported 25 CCHS patients in total comprising 10 patients with a combined CCHS/HSCR phenotype,^15,16,30,31 but RET germline mutations were reported solely in CCHS patients in association with HSCR. Amiel et al³¹ found a heterozygous P1039L mutation in a CCHS/HSCR patient who had inherited the mutation from the unaffected father, and Sakai et al¹⁵ also described a heterozygous T706A mutation in a CCHS/HSCR patient.¹⁵

We report a RET mutation in a patient with isolated CCHS/epilepsy phenotype. Although it was located in position ds+54 of intron 12, we did not find this variant in the normal control population. Intronic mutations close to this variant have been reported in association with HSCR.^28,44 Since the mutation derives from a C→A transversion, we hypothesise that it could generate a new splice acceptor site.

Furthermore, we describe a new P841L mutation in a CCHS/HSCR patient. Interestingly, the same amino acid change was found in codon 1039 of a CCHS/HSCR patient by Amiel et al.³¹ In addition, the substitution of a leucine for a proline amino acid residue was reported in association with isolated HSCR for codons 20, 64, 399, and 973.^24,26,44 In addition to the frequent occurrence of this substitution, all of these proline residues are conserved between humans and mice.

Besides the mutations, a rare RET variant of exon 18 codon 982 was found in a patient with combined CCHS and HSCR. This variant was not detected in our 312 control chromosomes, but Mulligan et al⁴⁰ found this polymorphism in three out of 142 normal chromosomes. Sancandi et al⁴⁵ detected the heterozygous codon 982 variant in four out of 57 sporadic HSCR patients⁴⁵ and Svensson et al²⁹ detected it in a sporadic HSCR patient who harbours an additional EDNRB mutation.²⁹ Although this variant is detected in controls and the phosphorylation activity of the RET molecule is not altered, it is considered not as a mutation but as a polymorphism.^42,43 However, the possibility remains that this polymorphism increases the risk of developing the HSCR or CCHS/HSCR phenotype by interfering with RET expression rather than with RET function.

We also found an association of a polymorphic RET variant with idiopathic congenital central hypoventilation syndrome. In particular, the c.135A variant is significantly over-represented in our CCHS population compared to normal controls. Although the statistical significance of this association is weak, this finding agrees with the strong association of this variant with the HSCR phenotype, as previously reported.^38,46 Interestingly, none of the c.2071G/A, c.2307T/G, or c.2712C/G RET polymorphic variants associated with the HSCR (particularly the c.2307G variant) were over- or under-represented in the CCHS population. In contrast, we found a tendency towards an over-representation of the c.1296A variant in our CCHS population. Analysing the allele frequencies of the c.1296G/A and c.135G/A polymorphisms in the isolated CCHS subpopulation and the CCHS/HSCR subpopulation, we found almost the same frequencies in both subpopulations for the c.1296G/A alleles (30 c.1296G and 20 c.1296A alleles in the isolated CCHS subpopulation and 10 c.1296G and six c.1296A alleles in the CCHS/HSCR subpopulation (χ²=0.032, p=1.000)). Further, the c.135A variant is slightly over-represented in the CCHS/HSCR subpopulation compared to the isolated CCHS subpopulation, albeit not significantly (33 c.135G and 17 c.135A alleles in the isolated CCHS subpopulation and nine c.135G and seven c.135A alleles in the CCHS/HSCR subpopulation (χ²=0.498, p=0.556)). Additionally, if compared to the normal controls, c.135A showed no statistically significant association with the isolated CCHS population (33 c.135G and 17 c.135A alleles in the isolated CCHS subpopulation and 238 c.135G and 74 c.135A alleles in the control population (χ²=2.421, p=0.159)). In spite of the limited sample size and absence of ANS dysfunction in controls and their families, these findings support the hypothesis of an involvement of specific RET haplotypes in the aetiology of CCHS and HSCR.

Finally, the identification of additional RET germline mutations in CCHS patients and the association of a specific RET variant with this phenotype suggest a contribution of RET to the aetiology of CCHS as the most severe form of ANS dysfunction. However, the low mutation rate, the incomplete penetrance, the variable phenotype, and the involvement of other genes like GDNF, EDN3, or BDNF further suggest that additional unidentified genes may be present, involved in the aetiology of idiopathic congenital central hypoventilation syndrome in a complex multigenic pattern of inheritance, in which a cumulative effect of mutations in multiple genes contributes to the phenotype.