The Research About Clinical Feature And Gene Mutation Detection In A Pedigree With Pulmonary Alveolar Microlithiasis

Pulmonary alveolar microlithiasis (PAM) is a rare autosomal recessive disease characterized by diffuse accumulation of innumerable spheric calcium phosphate microliths in the alveoli. Clinical-radiological dissociation is an important hallmark of this disease. Most PAM patients are asymptomatic and pulmonary tissue changes are discovered incidentally. Generally, PAM patients began to present symptoms mainly including repeated cough or progressive dyspnea. PAM has sporadic and familial occurrence and38-61%of PAM patients are familial.PAM is mainly pathologically attributed to formation and aggregation of calcium phosphate microliths in the alveoli after mutations in the SLC34A2gene (the type Ⅱb sodium-phosphate cotransporter gene) coding NaPi-Ⅱ b. As a member of solute-transfer protein family SLC34, SLC34A2widely expresses in multiple organic tissues and plays a crucial role in maintaining domestic inorganic phosphorus balance. In the clinical work, we discovered an inbred pedigree with PAM with four sibling PAM patients for clinical characteristics analysis, pathological genes test which contributes to research upon pathological mechanism, genetic characteristics and therapeutic guidelines of PAM.A pedigree with12members of3generations deriving from one first-cousin marriage was included in this study. Both founders of this pedigree were alive. They are blessed with four direct offsprings,3male and1female, all of which are diagnosed with PAM via radiological and pathological examinations and two (1male/1female) have deceased. The founders and their two granddaughters and one grandson were exempt from PAM after chest radiography. This pedigree is tagged as familial aggregation and steady inheritance thus suitable for study of PAM pathological mechanism.MATERIALS AND METHODSClinical data and characteristics of four patients are detailed and analyzed in the first part of this paper. The second part of this paper aimed at analyzing experimental process:5mL of peripheral blood was taken from nine family members respectively and kept under anti-coagulation with EDTA. Simultaneously, equal amount of peripheral blood was taken from10unrelated healthy subjects after physical examination. SLC34A2gene, equipped with13exons including the first non-coding exon and the rest12coding exons, is located on the short arm of chromosome4(4p15.1-p15.3). PCR primers were designed to amplify these12coding exons for amplication, including the exon sequence and a part of intron sequence (i.e.,5’and3’ flanking regions). PCR products sequencing were assigned to BGI Co., Ltd., Beijing, China. Sequence analysis was performed through the use of BioEdit Sequence Alignment Editor and a BLAST contrast was performed on NCBI. The third part of this paper, PAM pulmonary specimen acquired from percutaneous biopsy was compared with normal pulmonary tissue for studies of SLC34A2function. Firstly, SLC34A2eukaryotic expression vector was constructed by SLC34A2gene-implanted plasmid vector pcDNA3.1(+) via restriction enzymes Kpn I and Not I, then the recombinant plasmid was transformed into E. coli DH5a competent cells for restriction enzyme digestion and nucleotide sequencing analysis Once the pcDNA3.1(+)-SLC34A2recombinant plasmid was constructed successfully, A549cells resembling normal type Ⅱ alveolar epithelial cells in secreting pulmonary surfactant human lung adenocarcinoma cell were selected for transfection. PcDNA3.1(+)-SLC34A2recombinant transfer vector was transfected into A549cells by Lipofectamine2000cationic liposome for RNA extraction and reevaluation of recombinant plasmid. In both control group and PAM group, amplification fragments obtained form the locus at2000bp were in accordance with the size of expected SLC34A2fragment. In this way, pcDNA3.1(+)-SLC34A2recombinant transfer vector was totally expressed in A549cells. Cell culture supernatants from plasmid control group, normal control group and PAM group were collected and analyzed via automatic biochemical analyzer for phosphorus concentration48hours after successful transfection.Results:In total,3basic mutations at2exons were proved. A homozygous mutation (c.575C＞A) in exon6was discovered in family members202,203and207, with threonine to lysine mutation (p.T192K), belonging to amino acid substitution. A heterozygous mutation in exon6was discovered in family members101,102,301,302,303. The sequence of family member204and10healthy subjects was normal. Exon13sequencing was performed in all family members of this PAM pedigree and two nucleotide mutations were discovered. One mutation was a homozygous single-nucleotide polymorphism (SNP)(c.1901A>G) in exon13, which was discovered in all family members of this PAM pedigree and was rs6448389in NCBI SNP database. The other one was a homozygous mutation (c.1962G>A) in exon13, which was discovered in family members101,102,202,203,207and303, and a heterozygous mutation (c.1962G>A) in exon13, which was discovered in family members301and302. The exon13sequence was normal in family member204and unrelated healthy controls. This mutation changes from GGG to GGA. However, there were no changes in amino acids because both of them can code amino acids.Inorganic phosphorus concentration of cell culture supernatant decreased significantly in normal control group in comparison between plasmid control group (P<0.01) and PAM group(P<0.01). Inorganic phosphorus concentration of cell culture supernatant decreased slightly in PAM group yet still significantly in comparison with plasmid control group(P<0.01). The results confirmed that NaPi-Ⅱ b disfunction derived from mutated code of lysine rather than threonine owning to c.575C>Ahomozygous mutation at6th exon of SLC34A2.Conclusion:As a pedigree fell prey to autosomal-recessive inherited PAM with insidious onset, PAM is roughly diagnosed under general chest radiography. C.575C＞A homozygous mutation at6th exon of SLC34A2was confirmed according to gene sequencing which incurs miss-code of lysine rather than threonine or so called phenotype-genotype separation mutation. In correspondence to the genetic characteristics of this pedigree, this mutation is pertained as the genetic foundation of the pathogenesis of this PAM pedigree. In construction of neucleotide vector of SLC34A2gene and transfection of pulmonary epithelial cells, c.575C＞A homozygous at6th exon contributes to dysfunction of NaPi-Ⅱ b which incurs impaired phosphate transferring and decreased depletion of phosphorus ion ensuing accumulation and sediment of phosphate. Accordingly, calcium ions combine phosphates and finally form calcium phosphate (microlithiasis), leading to disease.