Download Molecular genetics of Polycystic Kidney Diseases

Molecular genetics of Polycystic Kidney Diseases
Eliecer Coto García
Laboratorio de Genética Molecular-Hospital Universitario Central de
Asturias (Oviedo), e Instituto de Estudios Nefrológicos (Fundación
Renal Iñigo Alvarez de Toledo)
Polycystic kidney diseases are a group of hereditary renal disorders with a familial
dominant (Autosomal dominant polycystic kidney disease, ADPKD) or recessive
(Autosomal recessive polycystic kidney disease, ARPKD) inheritance. Families with
ADPKD have mutations in either PKD1 (chromosome 16) or PKD2 (chromosome 4)
genes. PKD1 and PKD2 encode the polycystins,. ARPKD is the consequence of
mutations in a single gene on chromosome 6, PKHD1.
In ADPKD, mutations in PKD1 have been associated with more severe forms compared
to PKD2: significant younger age for End Stage Renal Disease (ESRD) and higher
rates of morbi-mortality. In this way, ADPKD2 is considered a benign form compared
to ADPKD1. Approximatelly, 90% of the ADPKD-families have mutations in PKD1.
The search for mutations in these families showed a wide spectrum of mutations, with
most of them being private mutations (specific for each family). DNA sequencing of
the PKD1 and PKD2 genes is difficult because the two are large genes, and mutations
are distributed in all the exons. This search for mutations could be useful for diagnostic
purposes, to define the type of disease (ADPKD1 vs. ADPKD2), but is not performed
for prenatal diagnosis, except for families with a high risk of cerebral aneurisms.
Because most of the mutation carriers (either PKD1 and PKD2) have echographic cysts
by the third decade of life, ultrasonographic analysis is highly accurate to define the
disease status of asymptomatic individuals from affected families.
ARPKD is a recessive disease with an early manifestation, including in utero. Patients
with severe forms carry two “severe” mutations (the parents being asymptomatic
carriers), and die in the perinatal period. Other patients carry at least one “non-severe”
mutation, and the disease manifests as a less severe phenotype. The PKHD gene
encodes fibrocystin, and is a large gene (>60 exons) making difficult the search for
mutations in these patients. However, some mutations have been found in several
patients, and the detection of a limited number of selected mutations would permit the
molecular diagnosis in approximately 80% of the patients. The prenatal diagnosis is
possible when the two mutations are characterised in one family.
Because the three are large genes and the mutations are randomly distributed, a
complete sequencing is required for each patient to define the mutations responsible for
the disease. This means that the genetic screening for these diseases is only affordable
for a few laboratories worldwide, and is very expensive in economic terms. It is
however possible an indirect approach, in which the chromosome that transmits with the
disease (or the two chromosomes in ARPKD) are identified by using microsatellite
polymorphisms. In this way, the family is characterised as ADPKD1 if all the patients
share the same 16-chromosome, and ADPKD2 if all carry the same chromosome 4.
This linkage approach requires the genotyping of at least five affected members in each
family, and this could be a limitation for most of the patients interested in a genetic
counseiling. In the case of ARPKD, the indirect approach requires that the phenotype of
the patient is clearly stablished as polycystic kidney disease, and both parents are free of
cysts. In this case, comparing the alleles for the microsatellites that the patient receive
from the mother and the father makes possible to define the two chromosomes 6 that
segregate with the disease.
The polycystins-1 and 2 and the fibrocystin are expressed in the primary cilia of
epithelial kidney cells, and the disfunction introduced by the mutated proteins is
traduced in characteristic cellular phenotypic changes (compared to normal non-mutated
cells): Renal cysts in epithelial cells, as a consequence of cellular de-diferenciation,
with excessive fluid secretion, and proliferation; alteration of cellular pathways such as
cilia, calcium homeostasis, cAMP, RAS/MAPK, and concentration capacity.
There are several polycystic mice and rat models, created by the disruption of several
genes, or the introduction of specific mutations in these genes. These animal models
have been used to test specific therapies, some of them under preclinical trials in
humans:
antiproliferative agents (such as targets of mTOR) and antagonists of
vasopressin V2 receptors. All these drugs are currently being tested for their capacity to
reduce the rate of cysts progression and increase the age of ESRD.
The next are questions frequently asked by the clinicians, in order to improve the
treatment of PKD patients or provide genetic counseiling:
-How could we differentiate the recessive from the adult form of PKD?.
-How and when a predictive test for adult PKD can be done?.
-What are the advantages of the molecular analysis for adult PKD, compared to
ultrasound examination?.
-When a prenatal test could be recommended in a family with a history of PKD?.
-Are there treatments for these diseases?.
-How to proceed with a genetic counseiling in the recessive form of PKD (including
prenatal diagnostics)?.
References.
Related Articles, Grantham JJ, Chapman AB, Torres VE.
Volume progression in autosomal dominant polycystic kidney disease: the major factor
determining clinical outcomes.
Clin J Am Soc Nephrol. 2006 Jan;1(1):148-57. Review.
Related Articles, Rossetti S, Consugar MB, Chapman AB, Torres VE, Guay-Woodford
LM, Grantham JJ, Bennett WM, Meyers CM, Walker DL, Bae K, Zhang QJ, Thompson
PA, Miller JP, Harris PC; CRISP Consortium.
Comprehensive molecular diagnostics in autosomal dominant polycystic kidney disease.
J Am Soc Nephrol. 2007 Jul;18(7):2143-60.
Torres VE, Harris PC, Pirson Y.
Autosomal dominant polycystic kidney disease.
Lancet. 2007 Apr 14;369(9569):1287-301. Review.
Related Articles,Torres VE, Harris PC.
Mechanisms of Disease: autosomal dominant and recessive polycystic kidney diseases.
Nat Clin Pract Nephrol. 2006 Jan;2(1):40-55. Review.
POLIQUISTOSIS RENALES HEREDITARIAS
Aspectos genético-moleculares
y
Nuevas posibilidades terapeuticas
ELIECER COTO GARCIA
Genética Molecular, Hospital Central de Asturias
Fundación Renal I. Alvarez de Toledo
Torres and Harris, J. Int. Med., 2007; 261:17-31
RESULTADOS
Estudio genético de SCN5A:
Mutación intrón 18 IVS -1G>A (1/9)
Polimorfismo H558R (2/9)
Antecedentes familiares:
Abuela fallecida mientras dormía
(+) 66 a
DAI
Hermano fallecido de muerte súbita a los 20 años
Padre diagnosticado de S. de Brugada y portador de DAI
ECG: Tipo II
34 a
20 a
MS
Test de flecainida positivo
(+) 29 a
EEF: negativo
(-) 5 a
Caso 4:FAMILIA PKD1
PKD1 (Arg871>Stop)
I
●
C/D
(+) 85 a A/B
II
●
A/C
●
(+)
50 a
(+)
45 a
ATGCGAGAGAGAGAn…GTGGTGA
n=7,A
n=8,B
n=9,C
n=10, D
…….
n=13,G
N=13,H
A/D
G/H
●
III
(+) 17 a
A/F
(-)
31 a
C/H
(+)
30 a
A/H
(-)
23 a
C/G
Torres and Harris, J. Int. Med., 2007; 261:17-31
mTOR: mammalian
Target of Rapamicina
Torres and Harris,
J. Int. Med., 2007;
261:17-31
Antagonistas del V2R (V2RA)
Las células quísticas proliferan, secretan fluido y destruyen el tejido adyacente.
El fluido quístico contiene, entre otras, hormona antidiurética (ADH) y epidermal
growth factor (EGF). Hay una acumulación de cAMP en el citoplasma de estas
células.
ADH estimula la expresión de V2R. cAMP la de aquaporinas (el agua difunde hacia
el intersticio).
Los V2RA reducen el tamaño de los quistes y el volumen renal en ratas pck
(PKHD1) y pcy (NPHP3).
Tolvaptan en humanos: incrementa la diuresis (sed y poliuria) pero es bien
tolerado. Ensayo con 1.500 pacientes con síntomas incipientes de PKD (Tolvaptan
o placebo).
Medir mediante Resonancia Magnética (RMI): incremento del tamaño de los
quistes y del riñón en un periodo de 5 años.
Hay una relación directa entre el incremento del tamaño de los quistes y el renal
total (RMI) y la tasa de filtración glomerular, la hipertensión, y la albuminuria
(ensayo con 241 pacientes PKD).
POLIQUISTOSIS AUTOSÓMICA Y RECESIVA
Afecta a 1/10.00 – 1/30.000 recién nacidos
Múltiples quistes renales, frecuentemente quistes hepáticos.
Fenotipo de Potter en formas neonatales severas
Hay pacientes con formas muy severas y otros con formas
menos severas (juveniles)
En todo caso, herencia recesiva (padres sanos), a diferencia
de las formas dominantes del adulto (genes PKD1 y PKD2)
En todas las familias estudiadas se ha obtenido ligamiento
positivo al cromosoma 6p21
En la primavera de 2002, tras 10 años de trabajo, dos
grupos de EEUU caracterizaron al gen PKHD1
PKHD1
A partir de la secuencia del genoma de la rata se identificó un gen (Pck) que estaba
mutado en una línea con una forma de poliquistosis similar a la humana
Se comparó la secuencia del gen Pck con la de todos los genes humanos, disponible
desde el año 2001
El gen humano más parecido al Pck era uno que estaba en 6p21
Se secuenció ese gen humano en varios pacientes poliquísticos hallándose varias
mutaciones: ese era el gen PKHD1
PKHD1 se extiende por unas 500 mil bases del cromosoma 6p21
En al menos 66 exones codifica un ARNm de unas 16 kilobases
El gen PKHD1 codifica la fibrocistina, una proteína con una gran dominio
extracelular, de función desconocida (adhesión célula-célula?), pero muy
abundante en riñones, páncreas, e hígado
Mutación Met 822 Val
Met 822
Met822Val
Mutación Lys 247 Arg
Lys247
Lys247Arg
MUTACIONES EN PKHD1
Rossetti, Torra, Coto, Consugar, Kubly, Málaga, Navarro, El-Youssef, Torres, Harris
A complete mutation screen of PKHD1 in autosomal recessive polycystic kidney disease pedigrees
Kidney Int 2003.
Pacientes: 47 casos (familias) con forma clásica de ARPKD (neonatal o juvenil),
y 14 adultos con fibrosis hepática congénita (CHF) y/o enfermedad de caroli (CD).
Métodos: Se analizaron los 66 exones del gen PKHD1.
Resultados: 33 mutaciones, en 22 de los 47 pacientes con ARPKD se hallaron
las dos mutaciones.
Hallamos al menos una de las dos mutaciones en el 85% de los casos severos, en el
42% de los casos moderados, y el 32% de los adultos con CHF y/o CD.
En los niños españoles hay dos mutaciones frecuentes, que sólo han sido halladas
en pacientes españoles: 9689delA, 5895insA.
Cribado genético para 5 mutaciones: 40 pacientes de toda España.
No mutaciones: 21; 9689: 8; 5895: 4; T36M: 5; I222V: 4; 1529: 2
En 5 casos se hallaron las dos mutaciones.
DIAGNOSTICO PREIMPLANTACIONAL
Normal/∆F508
Normal/∆F508
A
Normal/Normal
implantación
B
C
Normal/∆F508
∆F508 /∆F508
No implantación
Current Clinical Trials for ARPKD Patients
Evaluation of ARPKD and Congenital Hepatic Fibrosis Observational
Sponsor: National Human Genome Research Institute
The National Human Genome Research Institute at the National Institutes of
Health wants to collect comprehensive date on kidney and liver disease in
ARPKD/CHF and follow patients over time to provide the groundwork for more
focused studies and novel therapeutic interventions. The protocol includes children and adults with a clinical diagnosis of
ARPKD/CHF.
Patients who have received a kidney or liver transplant and have stable graft
function without severe complications are eligible.
The study requires admission to the NIH Clinical Center for four to five days, with
follow-up visits every one to two years. This study is currently enrolling patients. Chronic Kidney Disease in Children Prospective Cohort Study (CKiD) Observational
Sponsors: National Institute of Diabetes and Digestive and Kidney Diseases
(NIDDK), National Institute of Neurological Disorders and Stroke (NINDS),
National Institute of Child Health and Human Development (NICHD) and
National Heart, Lung, and Blood Institute (NHLBI).
This is a prospective, epidemiological study of children with chronic kidney
disease and includes children with ARPKD and ADPKD. The primary goals are to determine the risk factors for decline in kidney
function and to define how a progressive decline in kidney function impacts
neurocongitive function and behavior; the risk factors for cardiovascular
disease; growth failure and its associated morbidity.
This study is currently enrolling patients. Estudio genético de PKD2:
Mutación intrón 8 IVS -1G>A
(+) 66 a
(-) 34 a
20 a
(+) 29 a
(-) 5 a