A recessive contiguous gene deletion of chromosome 2p16 associated with cystinuria and a mitochondrial disease

Abstract

Deletions ranging from 100 Kb to 1 Mb--too small to be detected under the microscope--may still involve dozens of genes, thus causing microdeletion syndromes. The vast majority of these syndromes are caused by haploinsufficiency of one or several genes and are transmitted as dominant traits. We identified seven patients originating from an extended family and presenting with a unique syndrome, inherited in a recessive mode, consisting of cystinuria, neonatal seizures, hypotonia, severe somatic and developmental delay, facial dysmorphism, and lactic acidemia. Reduced activity of all the respiratory chain enzymatic complexes that are encoded in the mitochondria was found in muscle biopsy specimens of the patients examined. The molecular basis of this disorder is a homozygous deletion of 179,311 bp on chromosome 2p16, which includes the type I cystinuria gene (SLC3A1), the protein phosphatase 2Cbeta gene (PP2Cbeta), an unidentified gene (KIAA0436), and several expressed sequence tags. The extent of the deletion suggests that this unique syndrome is related to the complete absence of these genes' products, one of which may be essential for the synthesis of mitochondrial encoded proteins.

Figure 1

a, Pedigree of the extended Bedouin family with seven children affected with cystinuria, neonatal seizures, hypotonia, severe somatic and developmental delay, facial dysmorphism, mitochondrial disease, and lactic acidemia. Family members whose symbols are numbered and marked with a small horizontal line were available for the DNA studies. b, Linkage analysis to markers D2S2306 and D2S177, showing one homozygous allele in all patients. The pedigree members' symbols, marked as in a, are arranged above each gel lane. Markers D2S119 and D2S2220 showed similar patterns.

Figure 2

a, Results of PCR amplifications of the SLC3A1 exons (1–10) and an unrelated gene (N.R.). The patients’ DNA (V10 and V15 in fig. 1a) failed to amplify the SLC3A1 exons, whereas good amplification was observed for DNA from a parent (IV1 in fig. 1a) and a control. The quality of the patients’ DNA was assessed by its ability to amplify an unrelated gene. A = affected, P = parent, C = unrelated control individual, and M = molecular weight marker X of Amersham. b, Multiplex PCR assay defining the deletion borders. Affected children are indicated by blackened symbols. The PCR reaction included primers a, b, and c, as indicated in fig. 3a. Primers’ concentrations were 1 mM for primers b and c and 0.5 mM for primer a; annealing temperature was 57°C. The 439-bp product was obtained only if a normal allele was present; the 376-bp product was obtained only if a deletion allele was present. All parents are therefore heterozygotes for the deletion and the wild-type allele. M = the molecular weight marker X of Amersham. c, Results of RT-PCR from lymphoblastoid RNA from an affected individual (A; V10 in fig. 1a) compared with control (C) of the SLC3A1, PP2Cβ, and KIAA0436 genes. None of these transcripts are PCR-amplified in the affected individual. The actin gene was included to assess RNA quality. Primers for the PCR were selected on different exons, to avoid amplification from residual DNA in the RNA preparation.

Figure 3

a, Schematic representation of the deleted region. Upper part, The published sequence of the clones complementary to the sequence reported here. Lower part, Details of the clones. The genes contained in the region are indicated by horizontal lines above them. Alignment of each exon was performed by means of the BLAST program of the genomic sequence to the nr database. The 10 exons of the SLC3A1 (gray squares) have been published elsewhere. The genomic organization of the PP2Cβ and KIAA0436 exons (black squares and squares marked with horizontal lines, respectively) was derived from comparison of the mRNA sequence with the genomic sequence. The numbers of the first and last exons of these genes are indicated below the ruler line. Note that exon 10 of SLC3A1 and exon 14 of KIAA0436 overlap; this overlap is in the 3′ untranslated regions of both genes. ESTs are represented by white squares. A hatched square between exons 4 and 5 of the PP2Cβ gene represents a clone, AL11753, associated with a UniGene cluster that demonstrates no open reading frame. A pseudoribosomal gene in the interval between SLC3A1 and PP2Cβ is shown by a gray square, and “ψ Ribosomal” appears below it. The positions of the primers used for the definition of the deletion (primers a and b) and for the detection of the wild-type allele (primer c) are marked by arrowheads. Light vertical lines represent the positions of STSs that were not amplified in the affected children. Bold vertical lines represent the positions of STSs that were amplified in the affected children. The numbers on the ruler line are the original numbers in the published sequence of the clones (as appearing in May 1, 2001). The numbers at the ends of clone RP11-24I5 that overlap the numbers of the overlapping clones are indicated. b, The sequence at the joining of the deletion ends. The PCR product derived from patient’s DNA with primers a and b (as indicated in a) was subjected to sequencing. The regions contained in the adjacent clones RP11-194L1 and RP11-559M23 are represented by the upper lines. The four bases TTCC could be derived from either clone, as they are present in both.