Genetic Variability of Surfactant Protein-B and Respiratory Distress Syndrome: Clinical Implications

, Departments of Cellular and Molecular Physiology and Pediatrics; , Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, Pa.

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In This Article

Molecular Characterization of the Human SP-B Gene

The Nature of Genes

A gene consists of a linear array of nucleotides (there are four possible nucleotide bases: adenine [A], guanine [G], cytosine [C], or thymine [T]) that in turn specify the sequence of amino acids comprising cellular proteins (ie, enzymes, hormones, structural proteins). Therefore, genes (and ultimately, the entire genetic constitution or genotype of an organism) determine the nature of proteins (or phenotypes, ie, the outward expression of the genotype as determined both genetically and environmentally) in a cell, organ, or organism. A gene is first transcribed in the nucleus into a complementary RNA molecule. This messenger RNA is then processed into mature RNA, which is transported to the cytoplasm to be translated into a specific protein on ribosomes.

In the 1970s, researchers showed that genes are not necessarily made up of a contiguous piece of DNA. The nucleotide sequence of a gene consists of coding (exons) and noncoding (introns) regions. The exons determine the amino acid sequence of the protein. The coding sequence can be interrupted by introns (Note: there are genes without introns). Although introns are present in the initial RNA transcript, they are removed by splicing before the "mature" RNA molecule is moved to the cytoplasm for translation into the protein product. The nucleotide sequence of a gene consists of regions that encode the primary structure (sequence of amino acids) of proteins (structural regions) and regions that regulate the expression of a given gene under various conditions (regulatory regions). The regulatory regions of a gene usually flank the gene, although regulatory elements located within introns have been identified. These either remain untranscribed or are transcribed into RNA (but not translated into protein). If the transcribed regulatory regions precede the gene, they are referred to as 5' untranslated regions (5'UTR), and if they follow the gene, they are referred to as 3' untranslated regions (3'UTR). Short DNA sequences that regulate expression of genes are called motifs. A motif can also describe a short DNA sequence of unknown function that is repeated.

Human SP-B Gene

The human SP-B gene is located on the short arm of chromosome 2[12] and consists of 11 exons.[13] Figure 2 shows the genomic structural organization of the human SP-B gene. The exons are shown as numbered boxes and the introns are represented by lines connecting these "exon boxes." Exon I contains a short 5'UTR. A portion of exon X and the entire exon XI contain the 3'UTR. Both the 5'UTR and 3'UTR are shown in yellow in Figure 2. The translation start site (ATG) in exon I and the translation stop site (TGA) in exon X are noted.

Human SP-B gene: from gene to mature protein. Exons are shown as numbered boxes (Roman).

From Gene to Mature Protein

The SP-B gene encodes a large precursor molecule (42kDa). This precursor[14] undergoes post-translational processing (reviewed in [15]) to produce the mature SP-B product (a small hydrophobic protein [8kDa]). The mature SP-B is encoded by exons VI and VII shown in orange in Figure 2, and contributes to the function of surfactant. Figure 2 also shows the SP-B RNA transcript, which corresponds to those genomic regions that are actually transcribed and are part of the mature RNA molecule which will direct the translation of SP-B precursor protein. The precursor protein (the translation product of the entire coding sequence) and the mature SP-B protein processed from this precursor (which is found in surfactant) are also depicted.

Genetic Heterogeneity of Human SP-B

In addition, Figure 2 schematically depicts different types of mutations/polymorphisms (variants) of the SP-B gene and their potential impact on SP-B function (all known variants of SP-B are shown in Figure 3; please note that these 14 variations are distributed over the entire SP-B gene -- 5' flanking region, 3' untranslated region, exons, and introns). A transmissible alteration in the nucleotide sequence of DNA is referred to as a mutation or polymorphism. If the change leads to the absence of protein or to a variant protein that is dysfunctional or nonfunctional, the nucleotide change is referred to as a mutation. If the nucleotide change does not alter the encoded amino acid or if it does alter the amino acid and the changed amino acid does not, under normal circumstances, result in protein dysfunction (and therefore no consequences clinically), the nucleotide change is referred to as a polymorphism. Each gene occupies a specific region on a chromosome (ie, a locus). Each such DNA region or locus is said to be polymorphic, meaning it contains more than one version of the gene if the frequency of the most rare variant or allele (a genetic variant of the same gene) for the given locus is 0.01, so that the frequency of the heterozygotes is at least 0.02.

Mutations/polymorphisms in human SP-B gene.

Nucleotide changes within the SP-B gene include mutations within coding regions that shift the reading frame and therefore the final amino acid sequence specified, as well as mutations/polymorphisms within introns or untranslated regions that have the potential to affect regulation and/or splicing efficiency. For example, one nucleotide deletion (1553delT) within exon IV changes the reading frame (ie, a frameshift mutation) at amino acid 122 (122delT -- shown in red in Figure 2) which in turn creates a premature termination codon (a codon is a triplet of nucleotides which specifies a single amino acid; a termination or stop codon is a sequence of three consecutive nucleotides which signals the termination of protein synthesis and the release of the growing polypeptide chain from the ribosome) at amino acid 214 in exon VI. Translation therefore occurs prematurely, resulting in the absence of mature SP-B.[16] Genes are read as a sequence of nucleotide triplets (codons); a single nucleotide deletion or insertion therefore "shifts" the reading frame, resulting in an alternate sequence of amino acids being read and translated into protein product. A missense mutation, which involves a nucleotide substitution that alters one or more codons so that different amino acids are specified, in exon VII, which is part of the mature SP-B -- 4380 C/T -- changes amino acid 236 from arginine to cysteine (Arg236Cys). Although this change is associated with partial SP-B deficiency, the mechanism through which this occurs is unknown.[17]

The first half of intron IV, starting at nucleotide 1968, contains a polymorphic region where several copies of a composite motif are found (shown as blue rectangles in Figure 2). This motif consists of a 20bp (that is, 20 base pairs of nucleotides) conserved segment followed by a variable number of CA repeats. Figure 2 shows variants of this polymorphic region: a normal allele with 11 motifs; a variant where 5 of the motifs are missing; and a variant with 3 additional motifs. SP-B variants have been found in higher frequency in the RDS population.[18] Although the gain or loss of these motifs has the potential to affect the efficiency of splicing and/or regulation of SP-B, at present there is no information available regarding these possibilities. A single base mutation/polymorphism at the 3'UTR in exon XI (4376 G/A) is described with no reported phenotype. These examples of mutations/polymorphisms are collectively indicative of the existence of several SP-B variants (alleles) and they also hold the potential for mediating the differences observed in regulation, function, and splicing efficiency among the various SP-B alleles. Such differences may in part determine clinical outcomes.

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