Abstract 339: Splice Acceptor Site Mutation in Cullin 3 is Causing Pseudohypoaldosteronism Type II.

A rare Mendelian form of hypertension is Pseudohypoaldosteronism type II (PHAII), also referred to as familial hyperkalemic hypertension (FHHt, OMIM 145260 ). Mutations in WNK1 and WNK4 , and the two recently identified genes KLHL3 and CUL3 have been reported as causes of this monogenic form of arterial hypertension. Mutations in WNK1 and WNK4 result in over-activity of the thiazide-sensitive Na-Cl cotransporter (NCC) in the distal convoluted tubule. In a Turkish family (parents and 5 children) one daughter was suspected of PHAII. This daughter has been diagnosed at age 24 years with severe hypertension (200/130 mm Hg), hyperkalemia (6.5 mmol/l; reference range 3.5-5.1), and normal renal function. Besides a short stature, no other phenotypical abnormalities were identified. The patient’s hypertension and hyperkalemia completely normalized with hydrochlorothiazide, 50 mg daily. All other family members are healthy. Sanger sequencing of WNK1 and WNK4 did not reveal any mutation. A de novo mutation in the daughter is the most likely cause of her disease. Full exome sequencing (Nimblegen SeqCapEZV2, Illumina HiSeq2000) of the seven family members identified 34,733 unique variants in our patient. By comparing the recent mutations reported by Boyden et al, we found an identical de novo mutation in CUL3 , a splice acceptor site mutation in intron 8. RT-PCR analysis of RNA extracted from fresh blood showed two cDNA species in the patient, corresponding to a normal sized CUL3 fragment and an abrogated variant. Sanger sequencing of this shorter form revealed a loss of all nucleotides of exon 9 excluding the first nucleotide and including the first nucleotide of exon 10. This results in an in-frame 57 amino acid deletion abrogating the function of CUL3. After 1 week withdrawal from diuretic medication, we found in the patient’s urinary exosomes, vesicles derived from renal epithelial cells, an increase in the abundance of phosphorylated (P)-NCC (active form) compared to urinary exosomes extracted from urine when using thiazide diuretics. This de novo splice acceptor site mutation found in a second patient with PHAII confirmed CUL3 as a cause of PHAII. This patient also showed up-regulation of P-NCC as reported in WNK1 and WNK4 mutations pointing to the same mechanism of FHHt.

A novel homozygous splice acceptor site mutation of KISS1R in two siblings with normosmic isolated hypogonadotropic hypogonadism

ContextLoss-of-function mutations of the kisspeptin-1 receptor gene, KISS1R, have been identified in patients with normosmic isolated hypogonadotropic hypogonadism (nIHH).ObjectiveTo investigate KISS1R defects in patients with absent or delayed puberty.PatientsWe investigated KISS1R gene defects in a cohort of 99 Brazilian patients with nIHH or constitutional delay of puberty (CDP).MethodsThe entire coding region of KISS1R was amplified by PCR followed by automatic sequencing. In addition, screening for KISS1R exonic deletions was performed by multiplex ligation-dependent probe amplification.ResultsOne novel homozygous KISS1R mutation was identified in two siblings with nIHH. This variant was an insertion/deletion (indel) mutation characterized by the deletion of three nucleotides (GCA) at position −2 to −4, and by the insertion of seven nucleotides (ACCGGCT) at the same position, within the 3′ splice acceptor site of intron 2 of KISS1R. The brothers who carried this KISS1R mutation had no clinical evidence of pubertal development at the ages of 14 and 20 years. Computational analysis of this indel mutation predicted the generation of an abnormal protein. In addition, a new heterozygous KISS1R variant (p.E252Q) was identified in a male patient with sporadic nIHH. However, in vitro studies of this variant did not demonstrate functional impairment. Only known polymorphisms were identified in patients with CDP.ConclusionLoss-of-function mutations of KISS1R represents a rare cause of nIHH, and was absent in patients with CDP. We have described a novel KISS1R homozygous splice acceptor site mutation in the familial form of nIHH.