Methods

Blood, and in some cases cultured skin fibroblasts were obtained from patients following informed written consent. In one case, only fetal skin and cartilage were available. In this particular case, and in many others, blood was also obtained from the parents. The diagnosis of CD was based on typical clinical, neuroradiologic and biochemical criteria. The clinical manifestations of the patients are summarized in Table 1. Their ethnic origins are included in tables 2 and 3.

Table 1. Clinical manifestations 38 non-Jewish CD patients

Manifestations n

Age of onset <2m 14

3-4m 20

Poor head control 38

Macrocephaly 38

Visual failure 38

White-matter disease 38

Seizures 13

Alive no 2

unknown 5

Procedures for cell culture, isolation of peripheral blood lymphocytes and extraction of total RNA and genomic DNA are described in Zeng et al. (2002). Genomic DNA was amplified using primers previously described (Kaul et al. 1993, 1994, 1996; Zeng et al., 2002). The procedure of Zeng et al. (2002) was also followed for the synthesis and amplification of cDNA. DNA and cDNA PCR products were either directly sequenced or cloned in pGEM-T Easy-Vector according to the manufacturer's suggested protocols and then sequenced. Automated sequencing was used.

In vitro mutagenesis, ASPA cDNA expression and assay of ASPA activity were performed as described by Zeng et al. (2002).

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