A splice site variant in MADD affects hormone expression in pancreatic β cells and pituitary gonadotropes
Kristiina Pulli, Jonna Saarimäki-Vire, Pekka Ahonen, Xiaonan Liu, Hazem Ibrahim, Vikash Chandra, Alice Santambrogio, Yafei Wang, Kirsi Vaaralahti, Anna-Pauliina Iivonen, Johanna Känsäkoski, Johanna Tommiska, Yasmine Kemkem, Markku Varjosalo, Sanna Vuoristo, Cynthia L. Andoniadou, Timo Otonkoski, Taneli Raivio
Kristiina Pulli, Jonna Saarimäki-Vire, Pekka Ahonen, Xiaonan Liu, Hazem Ibrahim, Vikash Chandra, Alice Santambrogio, Yafei Wang, Kirsi Vaaralahti, Anna-Pauliina Iivonen, Johanna Känsäkoski, Johanna Tommiska, Yasmine Kemkem, Markku Varjosalo, Sanna Vuoristo, Cynthia L. Andoniadou, Timo Otonkoski, Taneli Raivio
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Research Article Endocrinology Reproductive biology

A splice site variant in MADD affects hormone expression in pancreatic β cells and pituitary gonadotropes

Abstract

MAPK activating death domain (MADD) is a multifunctional protein regulating small GTPases RAB3 and RAB27, MAPK signaling, and cell survival. Polymorphisms in the MADD locus are associated with glycemic traits, but patients with biallelic variants in MADD manifest a complex syndrome affecting nervous, endocrine, exocrine, and hematological systems. We identified a homozygous splice site variant in MADD in 2 siblings with developmental delay, diabetes, congenital hypogonadotropic hypogonadism, and growth hormone deficiency. This variant led to skipping of exon 30 and in-frame deletion of 36 amino acids. To elucidate how this mutation causes pleiotropic endocrine phenotypes, we generated relevant cellular models with deletion of MADD exon 30 (dex30). We observed reduced numbers of β cells, decreased insulin content, and increased proinsulin-to-insulin ratio in dex30 human embryonic stem cell–derived pancreatic islets. Concordantly, dex30 led to decreased insulin expression in human β cell line EndoC-βH1. Furthermore, dex30 resulted in decreased luteinizing hormone expression in mouse pituitary gonadotrope cell line LβT2 but did not affect ontogeny of stem cell–derived GnRH neurons. Protein-protein interactions of wild-type and dex30 MADD revealed changes affecting multiple signaling pathways, while the GDP/GTP exchange activity of dex30 MADD remained intact. Our results suggest MADD-specific processes regulate hormone expression in pancreatic β cells and pituitary gonadotropes.

Authors

Kristiina Pulli, Jonna Saarimäki-Vire, Pekka Ahonen, Xiaonan Liu, Hazem Ibrahim, Vikash Chandra, Alice Santambrogio, Yafei Wang, Kirsi Vaaralahti, Anna-Pauliina Iivonen, Johanna Känsäkoski, Johanna Tommiska, Yasmine Kemkem, Markku Varjosalo, Sanna Vuoristo, Cynthia L. Andoniadou, Timo Otonkoski, Taneli Raivio

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Figure 3

Dex30 leads to reduced number of β cells, lowered insulin content, and impaired insulin processing in SC-islets.

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Dex30 leads to reduced number of β cells, lowered insulin content, and ...
(A) Schematic of SC-islet differentiation protocol. (B) PDX1, (C) NKX2-2, (D) SOX9, and (E) NKX6-1 mRNA levels in WT and dex30 cultures at stages 3–5, relative to undifferentiated stem cells (n = 4–6 for WT, n = 8–11 for dex30). (F) INS, (G) PCSK1, and (H) PCSK2 mRNA levels in WT and dex30 cultures at stages 6–7, relative to undifferentiated stem cells (n = 6 for WT, n = 9 for dex30 at ST6, n = 10 for WT, and n = 13–14 for dex30 at stage 7). (I) Total insulin and (J) proinsulin content in WT and dex30 SC-islets at stage 7 (n = 7–9 for WT, n = 9–12 for dex30). (K) Proinsulin-to-insulin ratio in WT and dex30 SC-islets at stage 7 (n = 7 for WT, n = 9 for dex30). (L) Static insulin secretion from WT and dex30 SC-islets at stage 7, in 2.8 mM glucose (G2.8), 16.8 mM glucose (G16.8), 16.8 mM glucose + 50 mM Exendin 4 (Ex4), and 2.8 mM glucose with 30 mM KCl (n = 5 for WT, n = 6 for dex30). (M) Dynamic insulin secretion in WT and dex30 SC-islets at stage 7. Conditions like in L (n = 7 for WT, n = 9 for dex30). (N) Area under curve quantification of dynamic insulin secretion in M. ***P < 0.001, **P < 0.01, and *P < 0.05 analyzed by Student’s t test (I–K and N) or multiple t tests (B–H and L).