because of variations in pathological and clinical features of the disease , npd is classified into those with deficiency of acid sphingomyelinase activity ( types a and b ) , and those with defective intracellular processing and transporting of ldl cholesterol , which is known as type c. however , npd type c ( npc ) is different at the biochemical and molecular levels with a higher incidence than npa and npb . both types
a and b are characterized by acid sphingomyelinase ( asm ) deficiency that leads to excessive sphingomyelin accumulation in all phagocytic cells and in neurons .
normally , the enzyme activity of asm leads to the breakdown of sphingomyelin into ceramide and phosphorylcholine in lysosomes .
generally , multiple organs such as spleen , liver , bone marrow , lymph nodes , lung and central nervous system are affected .
clinically , npa is distinguished by failure of thrive , hepatosplenomegaly and progressive neurodegeneration . at molecular genetics level ,
the smpd1 gene is approximately 5 kb long and the coding sequence is divided among six exons .
according to the human gene mutation database ( www.hmdb.cf.ac.uk ) , more than 100 different mutations ( including missense , nonsense , deletion , insertion , and splice site mutations ) have been reported in the smpd1 gene . to date
for instance , motamedi et al reported a case of npb in iran and described the clinical course of his patient .
furthermore , motamed et al reported a ten year study of liver biopsies in children 's medical center in tehran .
most identified patients were affected with npd and glycogen storage disease type 1 ( gsd - i ) .
in addition , majidzadeh et al reported a case of npd and compared clinical and molecular aspects of the disease .
hoshmand et al presented a novel mutation in the smpd1 gene ( data not published ) . here , we present the first molecular genetics diagnosis of npa in an individual from southwest iran .
a 2.5 year - old male , the first born child from first cousin parents was attending our center for genetic counseling and genetic diagnostics .
the patient showed typical signs of the niemann pick disease , such as hepatosplenomegaly , developmental delay , mental retardation , and foam cells in the bone marrow , hypotonia , and cherry red maculae .
unfortunately , measurement of the asm activity in the white blood cells failed and dna test was the only chance to establish a definite diagnosis . after obtaining informed consent ,
genomic dna was extracted from edta - anticoagulated whole blood by standard salting out procedures .
selective amplification of all 6 exons was performed in a volume of 25 l reaction containing 10 pmol of each primer ( tag copenhagen a / s , fruebjergvej3 , denmark ) and 50ng of genomic dna .
pcr was carried out by a set of designed primers using primer3out software ( http://frodo.wi.mit.edu/primer3/ ) .
exon 2 was amplified by two separate primer pairs to assist subsequent sequencing reactions temperature profile of reactions were as follow : initial denaturation at 95c for 3 min , 35 cycles of 95c for 30s , 56 - 60c for 30s and 72c for 45s and a final extension at 72c for 7 min .
direct sequencing of pcr products was carried out using abi automated sequencer 3700 according to the manufacture 's instruction ( abi 3700 , pe applied biosystems , foster city , ca , usa ) .
sequence analysis was performed with the software bioedit ( version 7.0.5.3 ) . according to the human reference sequence nm_000543.4 , a homozygous single guanine deletion , c.740delg ,
was observed in exon 2 of the smpd1 gene , which results in a premature stop codon ( ctg > tga ) between codons 256 - 257 ( p.gly247alafs*9 ) ( fig .
they are also obligate carrier . to confirm the pathogenic relevance of the detected deletion , 27 healthy individuals were tested .
chromatogram of sequencing reactions from a ) affected individual , b ) heterozygote parents , c ) healthy person . the location of the novel deletion is indicated with black arrow and the position of the resulting stop codon is shown in black circle on the chromatogram . at bottom ( d ) , partial sequence of the smpd1 gene with corresponding codons is illustrated .
to our knowledge by intensive literature searching , the present report demonstrates firstly the molecular genetics diagnosis of the npd in southwest iran . approximately 1 per 40,000 people of ashkenazi jewish descent has npa .
in contrast , there is no information about frequency and distribution of different types of npd in iran , particularly in southwest iran .
the disease refers to a group of disorders with deficiency in lipid storage , causing accumulation of fats in brain and liver that lead to serious damage or dysfunction of mentioned tissues . however , liver enlargement , brain damage , difficulty in walking , speaking and learning are characteristic symptoms for npa .
diagnosis of this type of disease is usually made by measuring the asm activity in white blood cells .
he was also referred to our molecular diagnostic center with the aim to find genetic cause of the disease .
the smpd1 gene is the only gene considered to be associated with npa . here , we report an unreported deletion of single guanine in exon 2 of the smpd1 gene , causing a truncated gene product .
exon 2 is unusually large , which encodes for 258 amino acids or approximately 44% of the entire asm polypeptide .
however , the pathogenic nature of the novel deletion was confirmed by screening of healthy individuals .
three common missense mutations account for more than 90% of the mutant alleles in individuals of ashkenazi jewish ancestry with npa .
but in contrast to the ashkenazi jewish population , most affected individuals with npa showed in previous studies unique mutations in the smpd1 gene . consequently , identification of new npa cases increases the probability to discover new specific mutations for the given population .
the present observations extend the mutation spectrum of the smpd1 gene in npa patients worldwide , and can be used for the prenatal or preimplatation genetic diagnosis , at least in southwest iran .
therefore , prevention seems to be more effective strategy in the next pregnancy for this family and other families with positive history .
 objectiveniemann pick disease ( npd ) type a ( npa : mim # 257200 ) is a lipid storage disorder with an autosomal recessive inheritance and occurrs by defect of the smpd1 gene encoding sphingomyelinase . 
 disruption of this enzyme leads to the accumulation of sphingomyelin in brain and liver , which in turn causes dysfunction or damage of tissue.methodswe report firstly a 2.5 year old boy with npa in southwest iran . 
 initially , the diagnosis was resulted on the basis of clinical symptoms . 
 the genomic dna of the suspected individual was subjected to exon sequencing of the smpd1 gene . according to the human reference sequence nm_000543.4 
 , a novel single guanine deletion resulting in a frameshift mutation ( p.gly247alafs*9 ) was observed in the smpd1 gene that might be causative for the outcome of the disease.findingsthe present report is the first molecular genetics diagnosis of the npa in southwest iran . 
 the detected deletion in the smpd1 gene is remarkable because of its novelty.conclusiondespite similar morbidity sga infants exhibited higher lethal complication rates following delayed meconium passage compared to aga infants . 