MATERIAL AND METHODS

Patients

We screened 28 HSCR patients, 18 sporadic and 10 familial male HSCR patients for whom we had previously not found a RET or GDNF mutation,²⁵ and two patients with both HSCR and hydrocephalus. Mutation analysis of EDN3 and EDNRB was not performed in all cases. In those cases in which the genes were screened, no mutation was found (unpublished data). The familial cases came from families with only male HSCR patients. Among the 18 sporadic cases, nine had long segment and nine short segment HSCR.

We also included two patients with a combination of HSCR and hydrocephalus. The first of these two patients is a 6 year old male, the second child of non-consanguineous, healthy, Spanish parents. Since the newborn period, he has had relative macrocephaly, with head circumference on the 50th centile, and weight and length below the 3rd centile. He has complete agenesis of the corpus callosum, adducted thumbs with camptodactyly of the remaining fingers, spastic paraplegia, especially of the legs, and hydrocephalus, together with Hirschsprung disease, pyeloectasia, and left testicular hypoplasia. He is mentally retarded without speech development, but has acceptable social contact and comprehension. The karyotype is normal, 46,XY, and cardiological and ophthalmological features were normal.

The second of these two patients was the second child of healthy, unrelated, Dutch parents. His older brother was normal. The boy was born by caesarean section after an uncomplicated pregnancy. Apgar scores were 7 and 8 at one and five minutes, respectively. Birth weight, length, and head circumference were within normal limits. After a period of severe and untreatable constipation, the diagnosis of Hirschsprung disease, short segment type, was made at the age of 3 months. In the first instance, a partial posterior myectomy of the rectal muscles was done, with insufficient result, followed by resection of the affected bowel segment. He had severe and long lasting postoperative urination problems. At 6 months, length and weight were on the 50th centile, whereas his head circumference was between the 75th and 90th centile. At 9 months, macrocrania was found to be progressive and the head circumference (49.5 cm) was now above the 97th centile. An MRI scan of the brain showed dilated cerebral ventricles, without structural brain anomalies. At 12 months, a ventriculoperitoneal drain was inserted. At 13 months, he was operated on for bilateral inguinal hernias. Pigment anomalies of the hair, which was uncombable, and diffuse alopecia was noted in the second year of life. Furthermore, he had a small depigmented area on his face and one on his lower left leg. His psychomotor development was normal with normal IQ test at 1 and 2 years of age. Karyotype was normal, 46,XY.

DNA analysis

To analyse the 28 exons of the L1CAM gene, we designed 28 amplicons, as we previously described.²⁶ Primers used for amplification, amplicon sizes, melting temperatures, and pools of PCR products used for loading on DGGE gels are listed in table 1. All fragments were amplified using a single PCR programme and DGGE analysis was performed under a single set of experimental conditions. Amplification was carried out in a 50 μl reaction mixture containing 100 ng genomic DNA, 0.25 mmol/l dNTP, 10 pmol of each primer, and 0.125 U Taq DNA polymerase. The PCR programme started with denaturation at 94°C for three minutes followed by five cycles of denaturation, one minute at 94°C, primer hybridisation, one minute at 56°C, and elongation, two minutes at 72°C, then by five cycles of one minute at 94°C, one minute at 53°C, two minutes at 72°C, and 25 cycles of one minute at 94°C, one minute at 5°C, and two minutes at 72°C. Eventually, an elongation step was added at 72°C for five minutes. PCR products of the patients were mixed with those of a control. The amount of control PCR products added was approximately half of the amount of patient PCR products. After mixing the PCR products, a heteroduplex step was performed: the samples were denatured at 96°C for 10 minutes followed by renaturation at 50°C for one hour. Subsequently, the PCR products were pooled. The pooled PCR products were applied to a 1 mm 9% polyacrylamide (PAA) gel (acrylamide:bisacrylamide 37.5:1) in 0.5 × TAE (1 × TAE = 40 mmol/l Tris, HAC pH 8.0, 20 mmol/l NaAc, 1 mmol/l Na₂EDTA) containing a denaturing gradient of 45-80% urea-formamide (100% urea-formamide (UF) contains 7 mol/l urea and 40% deionised formamide). Gels were run at 100 V/19 cm and 58°C for 16 hours. After staining the DNA with ethidium bromide, the gel pattern was documented. Using an ABI PRISM 377 DNA sequencer (Perkin Elmer), direct sequencing of independently amplified PCR products was performed in both sense and antisense directions, with the same primers as used for DGGE, but without GC clamp whenever an aberrant DGGE pattern was found.

RESULTS

Out of 30 patients examined only two L1CAM-DGGE variations were identified. In a long segment HSCR patient, we detected a C to T change (C362T), which did not result in an amino acid change (Ser120Ser) and was therefore considered a non-pathogenic variant. The second DGGE variant was present in one of the two patients with HSCR and MASA/hydrocephalus. Sequencing showed that a T to G tranversion caused the DGGE variant 6 bp after the splice donor site of exon 5 (IVS5+6 T>G). To test the possible splicing effect of this tranversion, we isolated RNA from lymphocytes of the patient. cDNA was made and a first PCR was performed using primers of exons 3 and 8 (L1Ffor 5`-TCACGGAAC AGTCTCCACGG-3` and L1Frev 5`TGATGGTGGGCGTGGGA AAG-3`). This gave a PCR product of 714 bp. A nested PCR reaction were performed with primers in exons 4 and 6 (L1Nfor 5`-TCACGGGCAACAACAGCAC-3` and L1Nrev 5`-ATGATGGTCCTGGTGCCTGG-3`). This gave a PCR product of 370 bp. The PCR product of the patient gave the expected 370 bp long PCR product. A normal sequence of the fragment was confirmed by sequencing, making this variant most likely not pathogenic.

DISCUSSION

A substantial role of the L1 protein in the development of HSCR explaining the excess of affected males is very unlikely, as we found no pathogenic mutations in 28 male HSCR patients. We cannot, however, totally exclude involvement of L1CAM, as we might have missed mutations by the DGGE system used or more likely missed mutations in the non-scanned regions of the gene; most of the intronic sequences were not screened nor were the regulatory sequences of the 3` and 5` untranslated regions of the gene. A third possibility, in particular for the two patients with the combined phenotype, might be involvement of another gene.