Distribución de los circuitos y cálculo de sus protecciones del Monasterio

Three patients, H6, H I5 and H3, showed a conformational change by radiolabelled SSCP analysis of the PCR-amplified fragment of exon 2 (Fig 3.11). In patients H3 and H6, the band shift was observed in the single strands while in patient H I5 a very subtle change was observed in the double strand and no changes were seen in the single strands. The remaining 28 patients analysed for this exon had band patterns identical to the normal controls suggesting that a sequence change was not present in this region of the IDS gene.

Patient H6

An A-> G transition at position 311 in the cDNA was detected after sequencing of

c

genomic DNA (Fig 3.12). This transition is predicted to cause a missense mutation by changing amino acid 63 from asparagine (AAT) to aspartic acid (GÀT) (N63D). T h i s mutation abolishes a restriction site for the enzyme Ssp I (AAT i ATT), which was used to confirm the mutation in the index case and for carrier detection in one family member. PCR- amplified DNA from the mutant allele remains undigested and a 220bp product is observed in agarose gel electrophoresis, while the amplified normal allele is digested to fragments of 116 and 104 bp. These fragments are not separated under the conditions used for the gel mix and are seen as a single thick band. The relative of patient H6 tested, a female cousin, was shown to be a carrier for the N63D mutation by the presence o f both the undigested and digested bands (Figure 3.13).

(a) 4

(b)

6 10 11

Figure 3.11 Radiolabelled SSCP analysis of PCR-amplified fragment from exon 2.

(a) Lane 1 is a normal control. Lanes 2-7 are patients with Hunter disease. Band shifts were detected in lanes 2 and 3 in DNA from patients H6 and H3 respectively.

(b) Lane 1, patient HIS. Lanes 2-8, other patients with Hunter disease. Lanes9-11, norm al controls.

Chapter 3. Mutation analysis in patients with Hunter disease

NORMAL CONTRO: PATIENT H6

A-> G

C T A G C T A G

Figure 3.12 Sequencing of amplified fragment from exon 2 from genomic DNA of patient H6

The A—►G transition in this patient is indicated by arrow

1 2 3 4 M

Figure 3.13 Agarose gel electrophoresis of PCR amplifîed fragment of exon 2 digested with Ssp I for detection of carriers for the mutation found in patient H6.

Lane 1, patient H6. Lane 2, female relative of patient H6. Lanes 3-4, normal controls. M, X 1 kb DNA size marker.

Patient H15

This is a female patient who is one of a pair o f identical twins. She was previously reported to show discordant expression of a mutant X-chromosome through nonrandom X- inactivation which was shown by Southern blotting using the M 27p probe. Her tw in sister had a normal pattern of X-inactivation and did not show clinical signs o f the disease (Winchester et ah, 1992). Here, she was further analysed at the DNA level. Sequencing o f an amplified fragment of genomic DNA from exon 2 showed a com plex band pattern from nucleotide 247 when compared to normal control (Figure 3.14) From this nucleotide position onwards, double bands were observed across the gel which was an indication that a deletion or insertion in the mutant X-chromosome has caused a frame shift. Consequently, heterozygosity was seen in each lane of the sequencing gel. It became clear, after comparison o f the two sequences, that the mutation in patient HI 5 is a deletion o f one nucleotide, C, at position 247. This mutation would be expected to cause a frameshift and to create a stop codon 18 amino acids downstream , within the same exon (LIIVDDLRPSLGCYGDKLV to LSSWMTCAPPWAVMGISWX) NORMAL CONTROL C T A G 5' 5' C C T T T C T T T C C T T T C C C T 4 - A A T T C C A A T T C C G G T T G G G G 3’ 3' PATIENT H15 A G

Figure 3.14 Sequencing of amplified fragment of exon 2 from genomic DNA from patient HIS

The normal sequence is shown in the left hand panel. The deletion point is indicated by an arrow and both sequences observed in patient's H I5 DNA are shown in the right hand panel.

Chapter J. Mutation analysis in patients with Hunter disease

This mutation abolishes a restricton site for the enzyme MslI (CAPyNNiNNPiiTG) and PCR o f exon 2 followed by digestion with this enzyme was used to analyse famiily members for the presence of the deletion found in the index case. A heterozygous pattern was observed for some of the individuals tested, including male relatives and an unaffected male control, and was an indication o f partial digestion rather than heterozygosity (Figure 3.15). The same analysis was repeated increasing the amount o f enzyme and decreasing the am ount o f PCR product used in the digestion and the same results w ere observed. Consequently, this enzyme has proven not to be efficient for carrier detection in this family and for that reason, sequencing o f all family members was performed. The results for the sequencing reactions are shown in Figure 3.16. The twin sister showed the sam e sequencing pattern observed in the index case, indicating a carrier status for the Ibp deletion. All other family members showed the same sequencing pattem observed in norm al controls, indicating that they were not carriers for the mutation found in patient HIS.

Figure 3.15 Agarose gel electrophoresis of amplified exon 2 followed by digestion with Msl I for detection of carriers for the mutation found in patient HIS.

Lane 1 is the patient's mother. Lanes 2 and 3 are patient H 15 and her twin sister. Lanes 4-7 are the father, the brother, one sister and one half sister. Lane 8 is a digested male control and Lane 9 is an undigested male control.

Pedigree o f family o f patient HI 5

6

5 7

3 4

2

Figure 3.16 Sequencing of amplified fragment from genomic DNA from exon 2 for detection of carriers for the mutation found in patient HIS.

The family members analysed are shown in the pedigree above and are indicated by the same numbers in the sequencing gels.

Chapter 3. Mutation analysis in patients with Hunter disease

Patient H3

A deletion of eight nucleotides from position 305 to 312 was identified by sequencing amplified exon 2 from genomic DNA from this patient (Figure 3.18). This deletion results in substitution of tyrosine for serine in the first affected codon, 61, and creates a stop signal in the next codon (SP to YX).

Prior to the publication of the intron/exon boundaries for the IDS gene RT-PCR of RNA from cultured fibroblasts from an aborted fetus from this family, which had been diagnosed enzymically as suffering from Hunter disease, was performed (Goldenfum et al., 1993). The entire coding region of the IDS gene was amplified as two overlaping fragments, 1 and 2 (Figure 2.2, Chapter 2). The mutation was detected initially by an altered restriction pattem for the Sau 961-digestion of cDNA (Figure 3.17a). It was predicted from the sizes of the restriction fragments and from inspection of the published cDNA sequence that this sequence change had affected the specific Sau 961 restriction site (Gi GNCC) at nucleotides 303-307. Therefore, primers were designed to amplify this region of the cDNA, fragment 3 (Figure 2.2, Chapter 2). The mutation was also confirmed by an altered mobility shift in SSCP (Figure 3.17b) and sequencing (Figure 3.18).

To show that the deletion was not an artifact of RT-PCR and selective cloning, the region containing the putative deletion was re-amplified independently from cDNA from the fetus and a normal control. The products were digested withy4/w I (AGiCT), which has restriction sites either side of the deletion. The resulting fragments were analysed by electrophoresis in a 13% non-denaturing polyacrylamide gel which will resolve bands differing by only a few bases. According to the cDNA sequence this digestion should yield 2 fragments of 118 and 111 bp in the control and of 118 and 103 in the mutant. The smaller band in the mutant clearly ran faster, consistent with a deletion of 8 bp (Figure 3.19).

338— 230— 165— — 230 (b) 1 2 3

.. J

Figure 3.17 (a) Digestion with Sau 961 of amplified cDNA fragment 1 from fetus H3

Lanes 1 and 2, normal controls. Lane 3, affected fetus. The normal cDNA was digested to 338, 230 and 165 bp fragments whereas the mutant cDNA was digested to 495 and 230 bp fragments

(b) SSCP analysis of amplified cDNA fragment 1 from fetus H3

Lanel, normal control. Lane 2, affected fetus. Lane 3, another Hunter patient. The altered mobility shift is indicated by arrow.

Chapter 3. Mutation analysis in patients with Hunter disease