Molecular Genetic Analysis Of A Chinese Family With Inherited Pituitary Dwarfism

Inherited Pituitary Dwarfism is a slow growth disease which caused by abnormality of hypothalamus or anterior pituitary so that the growth hormone's rhythm and amount of secretion is abnormal,or growth hormone receptor have defects,or the transmission of information after the receptor is blocked. Inherited Pituitary Dwarfism contains two types: one is Isolated Growth Hormone Deficiency (IGHD), the other one is Combined Pituitary Hormone Deficiency (CPHD), and IGHD contains four types: IGHD IA, IGHD IB,IGHD II and IGHD III. The therapy to the disease is mainly use growth hormone or growth hormone releasing hormone so far. Inherited Pituitary Dwarfism can be inherited in autosomal dominant, autosomal recessive, or X-linked patterns. However, autosomal recessive is the most common form of it. There are 17 genes which are known to be relevant to the disease.An Inherited Pituitary Dwarfism family which is inherited in autosomal recessive in Hubei province of China was identified. Linkage analysis to the 17 genes have shown the Growth hormone releasing hormone receptor (GHRHR) gene closely linked to this disease in the family. GHRHR gene has the most possibility to cause the Pituitary Dwarfism in the family. Mutational analysis was conducted for all exons and exon-intron boundaries of GHRHR (GenBank accession no.NC₀00007.12) using direct DNA sequence analysis by ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). The proband was chosen for mutation screening. We found a novel homozygosis mutation, a G to A transition located in the splice donor site at the beginning of intron 8 (IVS8+1G>A) when compared with the normal sequence, was also found in heterozygous fashion in the proband's mother and in her son. To confirm that IVS8+1G>A mutation is associated with the disease in the family, RLFP analysis carried out. The patients in the family showed homozygosis mutant allele, the carriers showed the presence of both wild type allele and mutant allele. RFLP analysis showed that the IVS8+1G>A mutation cosegregated with all affected individuals and ought to carriers in the family. No mutation was found in 50 normal chromosomes from the Chinese controls.To determine the consequences of the IVS8+1G>A mutation on the splicing of GHRHR transcripts, HeLa cells were transfected with expression vectors containing the normal or the mutant GHRHR minigene consisting of a genomic fragment spanning exon 7 to exon 9. RT-PCR amplification of GHRHR transcripts isolated from cells transfected with the wild-type minigene generated a major product consistent with normal splicing of introns 7, 8 and 9, whereas the IVS8+1G>A allele produced five products in patient, one kind has a delete of exon 8, the second, third and fouth have a 48bp,46bp and 34bp insert which come from intron 8-9, respectively, the fifth has a delete of 32bp which is belong to exon 8. No matter which alleles, if translated, these abnormal transcripts would give rise to truncated or frameshift leading to severely truncated proteins.The results show the splice mutation IVS8+1G→A of GHRHR gene is cause of Inherited Pituitary Dwarfism in the family.